Delta-12 desaturase gene suitable for altering levels of polyunsaturated fatty acids in oleaginous yeasts

ABSTRACT

The present invention relates to a Δ12 fatty acid desaturase able to catalyze the conversion of oleic acid to linoleic acid (LA; 18:2). Nucleic acid sequences encoding the desaturase, nucleic acid sequences that hybridize thereto, DNA constructs comprising the desaturase gene, and recombinant host microorganisms expressing increased levels of the desaturase are described. Methods of increasing production of specific ω-3 and/or ω-6 fatty acids are described by overexpression of the Δ12 fatty acid desaturase or by disruption of the native gene.

This application claims the benefit of U.S. Provisional Application No. 60/468,677, filed May 7, 2003, and U.S. Provisional Application No. 60/484,209, filed Jun. 30, 2003.

FIELD OF THE INVENTION

This invention is in the field of biotechnology. More specifically, this invention pertains to the identification of a nucleic acid fragment encoding a Δ12 fatty acid desaturase enzyme useful for disrupting or enhancing the production of polyunsaturated fatty acids (PUFAs) in oleaginous microorganisms, such as oleaginous yeasts.

BACKGROUND OF THE INVENTION

It has long been recognized that certain polyunsaturated fatty acids, or PUFAs, are important biological components of healthy cells. For example, such PUFAs are recognized as:

-   -   “Essential” fatty acids that can not be synthesized de novo in         mammals and instead must be obtained either in the diet or         derived by further desaturation and elongation of linoleic acid         (LA) or α-linolenic acid (ALA);     -   Constituents of plasma membranes of cells, where they may be         found in such forms as phospholipids or triglycerides;     -   Necessary for proper development, particularly in the developing         infant brain, and for tissue formation and repair; and,     -   Precursors to several biologically active eicosanoids of         importance in mammals, including prostacyclins, eicosanoids,         leukotrienes and prostaglandins.

In the 1970's, observations of Greenland Eskimos linked a low incidence of heart disease and a high intake of long-chain ω-3 PUFAs (Dyerberg, J. et al., Amer. J. Clin Nutr. 28:958-966 (1975); Dyerberg, J. et al., Lancet 2(8081):117-119 (Jul. 15, 1978)). More recent studies have confirmed the cardiovascular protective effects of ω-3 PUFAs (Shimokawa, H., World Rev Nutr Diet, 88:100-108 (2001); von Schacky, C., and Dyerberg, J., World Rev Nutr Diet, 88:90-99 (2001)). Further, it has been discovered that several disorders respond to treatment with ω-3 fatty acids, such as the rate of restenosis after angioplasty, symptoms of inflammation and rheumatoid arthritis, asthma, psoriasis and eczema. γ-linolenic acid (GLA, an ω-6 PUFA) has been shown to reduce increases in blood pressure associated with stress and to improve performance on arithmetic tests. GLA and dihomo-γ-linolenic acid (DGLA, another ω-6 PUFA) have been shown to inhibit platelet aggregation, cause vasodilation, lower cholesterol levels and inhibit proliferation of vessel wall smooth muscle and fibrous tissue (Brenner et al., Adv. Exp. Med. Biol. 83: 85-101 (1976)). Administration of GLA or DGLA, alone or in combination with eicosapentaenoic acid (EPA, an ω-3 PUFA), has been shown to reduce or prevent gastrointestinal bleeding and other side effects caused by non-steroidal anti-inflammatory drugs (U.S. Pat. No. 4,666,701). Further, GLA and DGLA have been shown to prevent or treat endometriosis and premenstrual syndrome (U.S. Pat. No. 4,758,592) and to treat myalgic encephalomyelitis and chronic fatigue after viral infections (U.S. Pat. No. 5,116,871). Other evidence indicates that PUFAs may be involved in the regulation of calcium metabolism, suggesting that they may be useful in the treatment or prevention of osteoporosis and kidney or urinary tract stones. Finally, PUFAs can be used in the treatment of cancer and diabetes (U.S. Pat. No. 4,826,877; Horrobin et al., Am. J. Clin. Nutr. 57 (Suppl.): 732S-737S (1993)).

PUFAs are generally divided into two major classes (consisting of the ω-6 and the ω-3 fatty acids) that are derived by desaturation and elongation of the essential fatty acids, LA and ALA, respectively. Despite a variety of commercial sources of PUFAs from natural sources [e.g., seeds of evening primrose, borage and black currants; filamentous fungi (Mortierella), Porphyridium (red alga), fish oils and marine plankton (Cyclotella, Nitzschia, Crypthecodinium)], there are several disadvantages associated with these methods of production. First, natural sources such as fish and plants tend to have highly heterogeneous oil compositions. The oils obtained from these sources therefore can require extensive purification to separate or enrich one or more of the desired PUFAs. Natural sources are also subject to uncontrollable fluctuations in availability (e.g., due to weather, disease, or over-fishing in the case of fish stocks); and, crops that produce PUFAs often are not competitive economically with hybrid crops developed for food production. Large-scale fermentation of some organisms that naturally produce PUFAs (e.g., Porphyridium, Mortierella) can also be expensive and/or difficult to cultivate on a commercial scale.

As a result of the limitations described above, extensive work has been conducted toward: 1.) the development of recombinant sources of PUFAs that are easy to produce commercially; and 2.) modification of fatty acid biosynthetic pathways, to enable production of desired PUFAs. For example, advances in the isolation, cloning and manipulation of fatty acid desaturase and elongase genes from various organisms have been made over the last several years. Knowledge of these gene sequences offers the prospect of producing a desired fatty acid and/or fatty acid composition in novel host organisms that do not naturally produce PUFAs. The literature reports a number of examples in Saccharomyces cerevisiae, such as:

-   -   Domergue, F., et al. (Eur. J. Biochem. 269:4105-4113 (2002)),         wherein two desaturases from the marine diatom Phaeodactylum         tricornutum were cloned into S. cerevisiae, leading to the         production of EPA;     -   Beaudoin F., et al. (Proc. Natl. Acad. Sci. U.S.A.         97(12):6421-6426 (2000)), wherein the ω-3 and ω-6 PUF-A         biosynthetic pathways were reconstituted in S. cerevisiae, using         genes from Caenorhabditis elegans;     -   Dyer, J. M. et al. (Appl. Eniv. Microbiol., 59:224-230 (2002)),         wherein plant fatty acid desaturases (FAD2 and FAD3) were         expressed in S. cerevisiae, leading to the production of ALA;         and,     -   U.S. Pat. No. 6,136,574 (Knutzon et al., Abbott Laboratories),         wherein one desaturase from Brassica napus and two desaturases         from the fungus Mortierella alpina were cloned into S.         cerevisiae, leading to the production of LA, GLA, ALA and STA.         There remains a need, however, for an appropriate microbial         system in which these types of genes can be expressed to provide         for economical production of commercial quantities of one or         more PUFAs. Additionally, a need exists for oils enriched in         specific PUFAs, notably EPA and DHA.

One class or microorganisms that has not been previously examined as a production platform for PUFAs are the oleaginous yeasts. These organisms can accumulate oil up to 80% of their dry cell weight. The technology for growing oleaginous yeast with high oil content is well developed (for example, see EP 0 005 277B1; Ratledge, C., Prog. Ind. Microbiol. 16:119-206 (1982)) and may offer a cost advantage compared to commercial micro-algae fermentation for production of ω-3 or ω-6 PUFAs. Whole yeast cells may also represent a convenient way of encapsulating ω-3 or ω-6 PUFA-enriched oils for use in functional foods and animal feed supplements.

Despite the advantages noted above, most oleaginous yeast are naturally deficient in ω-6 and ω-3 PUFAs, since naturally produced PUFAs in these organisms are usually limited to 18:2 fatty acids (and less commonly, 18:3 fatty acids). Thus, the problem to be solved is to develop an oleaginous yeast that accumulates oils enriched in ω-3 and/or ω-6 fatty acids. Toward this end, it is not only necessary to introduce the required desaturases and elongases that allow for the synthesis and accumulation of ω-3 and/or ω-6 fatty acids in oleaginous yeasts, but also to increase the availability of the 18:2 substrate (i.e., LA). Generally, the availability of this substrate is controlled by the activity of Δ12 desaturases that catalyze the conversion of oleic acid to LA.

There are a variety of known Δ12 desaturases disclosed in the public literature, some of which originate from fungal sources (e.g., Mortierella alpina, Emericella nidulans, Mucor rouxil). These desaturases are not known to be effective for altering fatty acid composition in oleaginous yeasts and are not preferred for use in oleaginous yeasts. Thus, there is need for the identification and isolation of genes encoding Δ12 desaturases that will be suitable for expression in these particular host organisms for use in the production of PUFAs.

Applicants have solved the stated problem by isolating the gene encoding a Δ12 desaturase from the oleaginous yeast, —Yarrowia lipolytica.

SUMMARY OF THE INVENTION

The invention relates to a gene encoding a Δ12 desaturase enzyme isolated from Yarrowia useful for the manipulation of the biochemical pathway for the production of ω-3 and/or ω-6 fatty acids. Accordingly, the invention provides an isolated nucleic acid molecule encoding a Yarrowia Δ12 desaturase enzyme, selected from the group consisting of:

-   -   (a) an isolated nucleic acid molecule encoding the amino acid         sequence as set forth in SEQ ID NO:24;     -   (b) an isolated nucleic acid molecule that hybridizes with (a)         under the following hybridization conditions: 0.1×SSC, 0.1% SDS,         65° C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1%         SDS; or an isolated nucleic acid molecule that is complementary         to (a) or (b).

Additionally the invention provides transformed host cells comprising the nucleic-acid molecules of the invention, genetic chimera and polypeptides encoded by the same.

In an alternate embodiment the invention provides a method for the production of linoleic acid comprising:

-   -   a) providing a yeast comprising:         -   (i) a chimeric gene of the invention encoding a Δ12             desaturase polypeptide; and         -   (ii) a source of desaturase substrate consisting of oleic             acid;     -   b) growing the yeast of step (a) under conditions wherein the         gene encoding a Δ12 desaturase polypeptide is expressed and the         oleic acid is converted to linoleic acid; and     -   c) optionally recovering the linoleic acid of step (b).     -   In another embodiment the invention provides a method for         producing ω-3 fatty acids comprising:         -   a) engineering a microbial host cell comprising the             following elements:             -   (i) a disrupted endogenous gene encoding a Δ12                 desaturase polypeptide; and             -   (ii) genes encoding enzymes of the ω-3 fatty acid                 biosynthetic pathway; and         -   b) providing a source of desaturase substrate consisting of             α-linolenic acid;         -   c) growing the yeast of step (a) under conditions wherein             the genes of the ω-3 fatty acid biosynthetic pathway are             expressed, producing ω-3 fatty acids; and         -   d) optionally recovering the ω-3 fatty acids of step (c).

Similarly the invention provides a method for modulating the biosynthesis of ω-3 or ω-6 fatty acids in a host cell comprising:

-   -   a) providing a host cell comprising a functional ω-3/ω-6 fatty         acid biosynthetic pathway;     -   b) over-expressing a Δ12 desaturase gene in the host cell of         (a); whereby the biosynthesis of ω-3 or ω-6 fatty acids is         modulated.

In another embodiment the invention provides a method of obtaining a nucleic acid molecule encoding a Δ12 desaturase enzyme comprising:

-   -   (a) probing a genomic library with the nucleic acid molecule of         the invention;     -   (b) identifying a DNA clone that hybridizes with the nucleic         acid molecule of the invention; and     -   (c) sequencing the genomic fragment that comprises the clone         identified in step (b),

wherein the sequenced genomic fragment encodes a Δ12 desaturase enzyme.

Similarly the invention provides a method of obtaining a nucleic acid molecule encoding a Δ12 desaturase enzyme comprising:

-   -   (a) synthesizing at least one oligonucleotide primer         corresponding to a portion of the sequence as set forth in SEQ         ID NOs:23; and     -   (b) amplifying an insert present in a cloning vector using the         oligonucleotide primer of step (a);         wherein the amplified insert encodes a portion of an amino acid         sequence encoding a Δ12 desaturase enzyme.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE DESCRIPTIONS

FIG. 1 shows a schematic illustration of the biochemical mechanism for lipid accumulation in oleaginous yeast.

FIG. 2 illustrates the ω-3 and ω-6 fatty acid biosynthetic pathways.

FIG. 3 illustrates the construction of the plasmid vector pY5 for gene expression in Yarrowia lipolytica.

FIG. 4 illustrates the construction of plasmid vectors pY5-13 and pY5-4 for gene expression in Y. lipolytica.

FIG. 5 shows a pairwise comparison (% Identity) between and among different yeast and fungal Δ12 desaturase homologs using a ClustalW analysis (Megalign program of DNASTAR sofware).

FIG. 6 is a schematic presentation of the construction of intermediate vector pYZM5CHPPA.

FIG. 7 shows a comparison between the DNA sequence of the Saprolegnia diclina Δ17 desaturase gene- and the synthetic gene codon-optimized for expression in Y. lipolytica.

FIG. 8 illustrates the favored consensus sequences around the translation initiation codon ‘ATG’ in Y. lipolytica.

FIG. 9 illustrates the strategy for in vitro synthesis of the codon-optimized Δ17 desaturase gene.

FIG. 10 shows plasmids for expression of the synthetic codon-optimized and wildtype Δ17 desaturase genes in Y. lipolytica.

FIGS. 11A and 11B show the results of gas chromatographic analysis of fatty acids produced in Y. lipolytica transformed with the wildtype and synthetic codon-optimized Δ17 desaturase genes, respectively.

FIG. 12 is a schematic presentation of the construction of intermediate vector pY24-4.

FIG. 13 is a schematic presentation of the construction of intermediate vector pYZV16.

FIG. 14 is a schematic presentation of the construction of integration vector pYZM5EL6.

FIG. 15 is a schematic presentation of the construction of integration vectors pYZV5EL6 and pYZV5EL6/17.

The invention can be more fully understood from the following detailed description and the accompanying sequence descriptions, which form a part of this application.

The following sequences comply with 37 C.F.R. §1.821-1.825 (“Requirements for Patent Applications Containing Nucleotide Sequences and/or Amino Acid Sequence Disclosures—the Sequence Rules”) and are consistent with World Intellectual Property Organization (WIPO) Standard ST.25 (1998) and the sequence listing requirements of the EPO and PCT (Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of the Administrative Instructions). The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822.

SEQ ID NOs:1 and 2 correspond to primers TEF5′ and TEF3′, respectively, used to isolate the TEF promoter.

SEQ ID NOs:3 and 4 correspond to primers XPR5′ and XPR3′, respectively, used to isolate the XPR2 transcriptional terminator.

SEQ ID NOs:5-18 correspond to primers YL1, YL2, YL3, YL4, YL23, YL24, YL5, YL6, YL9, YL10, YL7, YL8, YL61 and YL62, respectively, used for plasmid construction.

SEQ ID NOs:19 and 21 are the degenerate primers identified as P73 and P76, respectively, used for the isolation of a Yarrowia lioplytica Δ12 desaturase gene.

SEQ ID NOs:20 and 22 are the amino acid consensus sequences that correspond to the degenerate primers P73 and P76, respectively.

SEQ ID NO:23 shows the DNA sequence of the Y. lipolytica Δ12 desaturase gene, while SEQ ID NO:24 shows the amino acid sequence of the Y. lipolytica Δ2 desaturase.

SEQ ID NOs:25-28 correspond to primers P99, P100, P101 and P102, respectively, used for targeted disruption of the Y. lipolytica Δ12 desaturase gene.

SEQ ID NOs:29-32 correspond to primers P119, P120, P121 and P122, respectively, used to screen for targeted integration of the disrupted Y. lipolytica Δ12 desaturase gene.

SEQ ID NOs:33 and 34 correspond to primers P147 and P148, respectively, used to amplify the full-length Y. lipolytica Δ12 desaturase gene.

SEQ ID NO:35 shows the DNA sequence of the Saprolegnia diclina Δ17 desaturase gene.

SEQ ID NO:36 shows the DNA sequence of the Mortierella alpina Δ6 desaturase gene, while SEQ ID NO:37 shows the amino acid sequence of the M. alpina Δ6 desaturase.

SEQ ID NO:38 shows the DNA sequence of the Mortierella alpina Δ5 desaturase gene, while SEQ ID NO:39 shows the amino acid sequence of the M. alpina Δ5 desaturase.

SEQ ID NOs:40 and 41 correspond to primers YL11 and YL12, respectively, used for amplifying the M. alpina Δ5 desaturase.

SEQ ID NOs:42 and 43 correspond to primers YL21A and YL22, respectively, used for amplifying the wild type S. diclina Δ17 desaturase.

SEQ ID NO:44 shows the DNA sequence of the Mortierella alpina high affinity elongase gene, while SEQ ID NO:45 shows the amino acid sequence of the M. alpina high affinity elongase.

SEQ ID NO:46 shows the DNA sequence of the synthetic Δ17 desaturase gene codon-optimized for expression in Yarrowia lipolytica, while SEQ ID NO:47 shows the corresponding amino acid sequence of the S. diclina Δ17 desaturase.

SEQ ID NOs:48-69 correspond to the 11 pairs of oligonucleotides that together comprise the entire codon-optimized coding region of the S. diclina Δ17 desaturase gene (e.g., D17-1A, D17-1B, D17-2A, D17-2B, D17-3A, D17-3B, D174A, D17-4B, D17-5A, D17-5B, D17-6A, D17-6B, D17-7A, D17-7B, D17-8A, D17-8B, D17-9A, D17-9B, D17-10A, D17-10B, D17-11A and D17-11B, respectively).

SEQ ID NOs:70-75 correspond to primers D17-1, D17-4R, D17-5, D17-8D, D17-8U and D17-11, respectively, used for PCR amplification during synthesis of the codon-optimized Δ17 desaturase gene.

SEQ ID NOs:76 and 77 correspond to primers YL53 and YL54, respectively, used for site-directed mutagenesis to generate pYSD17M.

SEQ ID NOs:78 and 79 correspond to primers KU5 and KU3, respectively, used for amplifying a 1.7 kB DNA fragment (SEQ ID NO:80; amino acid sequence provided as SEQ ID NO:81) containing the Yarrowia URA3 gene.

SEQ ID NOs:82 and 83 correspond to primers KI5 and KI3, respectively, used for amplifying a 1.1 kB DNA fragment (SEQ ID NO:84; amino acid sequence provided as SEQ ID NO:85) containing the conjugase gene of Impatients balsama.

SEQ ID NOs:86 and 87 correspond to primers KTI5 and KTI3, respectively, used for amplifying a 1.7 kB DNA fragment (SEQ ID NO:88; amino acid sequence provided as SEQ ID NO:89) containing a TEF::conjugase::XPR chimeric gene.

SEQ ID NOs:90 and 91 correspond to primers KH5 and KH3, respectively, used for amplifying a 1 kB DNA fragment (SEQ ID NO:92; amino acid sequence provided as SEQ ID NO:93) containing the E. coli hygromycin resistance gene.

SEQ ID NOs:94 and 95 correspond to primers KTH5 and KTH3, respectively, used for amplifying a 1.6 kB DNA fragment (SEQ ID NO:96; amino acid sequence provided as SEQ ID NO:97) containing the TEF::HPT::XPR fusion gene.

SEQ ID NOs:98 and 99 correspond to the 401 bp of 5′-sequence and 568 bp of 3′-sequence of the Yarrowia lipolytica URA3 gene, respectively, used to direct integration of expression cassettes into the Ura loci of the Yarrowia genome.

SEQ ID NOs:100-103 correspond to primers YL63, YL64, YL65 and YL66, respectively, used for site-directed mutagenesis to generate pY24-4.

SEQ ID NOs:104-107 correspond to primers YL81, YL82, YL83 and YL84, respectively, used for site-directed mutagenesis to generate pYZM5CH.

SEQ ID NOs:108 and 109 correspond to primers YL105 and YL106, respectively, used for site-directed mutagenesis to generate pYZM5CHPP.

SEQ ID NOs:110 and 111 correspond to primers YL119 and YL120, respectively, used for site-directed mutagenesis to generate pYZM5CHPPA.

SEQ ID NOs:112 and 113 correspond to primers YL121 and YL122, respectively, used for amplifying 440 bp of 5′-non-coding DNA sequence (SEQ ID NO:114) upstream from the Y. lipolylica URA3 gene.

SEQ ID NOs:115 and 116 correspond to primers YL114 and YL115, respectively, used for site-directed mutagenesis to generate pYZV5 and pYZV5P.

SEQ ID NO:117 corresponds to a 5.2 kB DNA fragment suitable for integration and expression of the Δ5 desaturase gene in the Y. lipolytica genome.

SEQ ID NOs:118 and 119 correspond to primers YL69 and YL70, respectively, used for site-directed mutagenesis to generate pY58BH.

SEQ ID NOs:120-123-correspond to primers YL77, YL78, YL79A and YL80A, respectively, used for site-directed mutagenesis to generate pY54PC.

SEQ ID NO:124 corresponds to a 8.9 kB DNA fragment suitable for integration and coordinate expression of the Δ6 desaturase, PUFA elongase and Δ5 desaturase genes in the Y. lipolytica genome.

SEQ ID NOs:125-128 correspond to primers YL101, YL102, YL103 and YL104, respectively, used for site-directed mutagenesis to generate pYSD17SPC.

SEQ ID NO:129 corresponds to a 10.3 kB DNA fragment suitable for integration and coordinate expression of the Δ6 desaturase, PUFA elongase, Δ5 desaturase and Δ17 desaturase genes in the Y. lipolytica genome.

SEQ ID NO:130 corresponds to the codon-optimized translation initiation site for genes optimally expressed in Yarrowia sp.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the subject invention, Applicants have isolated and confirmed the identity of a Yarrowia lipolytica gene encoding a Δ12 desaturase. Additionally, methods and compositions are provided which permit modification of the long chain polyunsaturated fatty acid (PUFA) content of oleaginous yeasts, such as Yarrowia lipolytica.

The invention relates to a new Δ12 desaturase enzyme and gene encoding the same that may be used for the manipulation of biochemical pathways for the production of healthful PUFAs. The subject invention finds many applications. PUFAs, or derivatives thereof, made by the methodology disclosed herein can be used as dietary substitutes, or supplements, particularly infant formulas, for patients undergoing intravenous feeding or for preventing or treating malnutrition. Alternatively, the purified PUFAs (or derivatives thereof) may be incorporated into cooking oils, fats or margarines formulated so that in normal use the recipient would receive the desired amount for dietary supplementation. The PUFAs may also be incorporated into infant formulas, nutritional supplements or other food products and may find use as anti-inflammatory or cholesterol lowering agents. Optionally, the compositions may be used for pharmaceutical use (human or veterinary). In this case, the PUFAs are generally administered orally but can be administered by any route by which they may be successfully absorbed, e.g., parenterally (e.g., subcutaneously, intramuscularly or intravenously), rectally, vaginally or topically (e.g., as a skin ointment or lotion).

Supplementation of humans or animals with PUFAs produced by recombinant means can result in increased levels of the added PUFAs, as well as their metabolic progeny. For example, treatment with arachidonic acid (ARA) can result not only in increased levels of ARA, but also downstream products of ARA such as prostaglandins. Complex regulatory mechanisms can make it desirable to combine various PUFAs, or add different conjugates of PUFAs, in order to prevent, control or overcome such mechanisms to achieve the desired levels of specific PUFAs in an individual.

Definitions

In this disclosure, a number of terms and abbreviations are used. The following definitions are provided.

“Open reading frame” is abbreviated ORF.

“Polymerase chain reaction” is abbreviated PCR.

“American Type Culture Collection” is abbreviated ATCC.

“Polyunsaturated fatty acid(s)” is abbreviated PUFA(s).

The term “fatty acids” refers to long chain aliphatic acids (alkanoic acids) of varying chain length, from about C₁₂ to C₂₂ (although both longer and shorter chain-length acids are known). The predominant chain lengths are between C₁₆ and C₂₂. The structure of a fatty acid is represented by a simple notation system of “X:Y”, where X is the total number of carbon (C) atoms in the particular fatty acid and Y is the number of double bonds.

Generally, fatty acids are classified as saturated or unsaturated. The term “saturated fatty acids” refers to those fatty acids that have no “double bonds” between their carbon backbone. In contrast, “unsaturated fatty acids” have “double bonds” along their carbon backbones (which are most commonly in the cis-configuration). “Monounsaturated fatty acids” have only one “double bond” along the carbon backbone (e.g., usually between the 9^(th) and 10^(th) carbon atom as for palmitoleic acid (16:1) and oleic acid (18:1)), while “polyunsaturated fatty acids” (or “PUFAs”) have at least two double bonds along the carbon backbone (e.g., between the 9^(th) and 10^(th), and 12^(th) and 13^(th) carbon atoms for linoleic acid (18:2); and between the 9^(th) and 10^(th), 12^(th) and 13^(th), and 15^(th) and 16^(th) for α-linolenic acid (18:3)).

“PUFAs” can be classified into two major families (depending on the position (n) of the first double bond nearest the methyl end of the fatty acid carbon chain). Thus, the “omega-6 fatty acids” (ω-6 or n-6) have the first unsaturated double bond six carbon atoms from the omega (methyl) end of the molecule and additionally have a total of two or more double bonds, with each subsequent unsaturation occuring 3 additional carbon atoms toward the carboxyl end of the molecule. In contrast, the “omega-3 fatty acids” ω-3 or n-3) have the first unsaturated double bond three carbon atoms away from the omega end of the molecule and additionally have a total of three or more double bonds, with each subsequent unsaturation occuring 3 additional carbon atoms toward the carboxyl end of the molecule.

For the purposes of the present disclosure, the omega-reference system will be used to indicate the number of carbons, the number of double bonds and the position of the double bond closest to the omega carbon, counting from the omega carbon (which is numbered 1 for this purpose). This nomenclature is shown below in Table 1, in the column titled “Shorthand Notation”. The remainder of the Table summarizes the common names of ω-3 and ω-6 fatty acids, the abbreviations that will be used throughout the specification and each compounds' chemical name. TABLE 1 Nomenclature Of Polyunsaturated Fatty Acids Shorthand Common Name Abbreviation Chemical Name Notation Linoleic LA cis-9,12-octadecadienoic 18:2 ω-6 γ-Linoleic GLA cis-6,9,12- 18:3 ω-6 octadecatrienoic Dihomo-γ- DGLA cis-8,11,14- 20:3 ω-6 Linoleic eicosatrienoic Arachidonic ARA cis-5,8,11,14- 20:4 ω-6 eicosatetraenoic α-Linolenic ALA cis-9,12,15- 18:3 ω-3 octadecatrienoic Stearidonic STA cis-6,9,12,15- 18:4 ω-3 octadecatetraenoic Eicosa- ETA cis-8,11,14,17- 20:4 ω-3 tetraenoic eicosatetraenoic Eicosa- EPA cis-5,8,11,14,17- 20:5 ω-3 pentaenoic eicosapentaenoic Docosa- DPA cis-7,10,13,16,19- 22:5 ω-3 pentaenoic docosapentaenoic Docosa- DHA cis-4,7,10,13,16,19- 22:6 ω-3 hexaenoic docosahexaenoic

The term “essential fatty acid” refers to a particular PUFA that an individual must ingest in order to survive, being unable to synthesize the particular essential fatty acid de novo. Linoleic (18:2, ω-6) and linolenic (18:3, ω-3) fatty acids are “essential fatty acids”, since humans cannot synthesize them and have to obtain them in their diet.

The term “fat” refers to a lipid substance that is solid at 25° C. and usually saturated.

The term “oil” refers to a lipid substance that is liquid at 25° C. and usually polyunsaturated. PUFAs are found in the oils of some algae, oleaginous yeasts and filamentous fungi. “Microbial oils” or “single cell oils” are those oils naturally produced by microorganisms during their lifespan. Such oils can contain long chain PUFAs.

The term “PUFA biosynthetic pathway enzyme” refers to any of the following enzymes (and genes which encode said enzymes) associated with the biosynthesis of a PUFA, including: a Δ4 desaturase, a Δ5 desaturase, a Δ6 desaturase, a Δ12 desaturase, a Δ15 desaturase, a Δ17 desaturase, a Δ9 desaturase and/or an elongase.

The term “ω-3/ω-6 fatty acid biosynthetic pathway” refers to a set of genes which, when expressed under the appropriate conditions encode enzymes that catalyze the production of either or both ω-3 and ω-6 fatty acids. Typically the genes involved in the ω-3/ω-6 fatty acid biosynthetic pathway encode some or all of the following enzymes:Δ12 desaturase, Δ6 desaturase, elongase, Δ5 desaturase, Δ17 desaturase, Δ15 desaturase, Δ9 desaturase and Δ4 desaturase. A representative pathway is illustrated in FIG. 2, providing for the conversion of oleic acid through various intermediates to DHA, which demonstrates how both ω-3 and ω-6 fatty acids may be produced from a common source. The pathway is naturally divided into two portions where one portion will generate ω-3 fatty acids and the other portion, only ω-6 fatty acids. That portion that only generates ω-3 fatty acids will be referred to herein as the ω-3 fatty acid biosynthetic pathway whereas that portion that generates only ω-6 fatty acids will be referred to herein as the ω-6 fatty acid biosynthetic pathway.

The term “functional” as used herein in context with the ω-3/ω-6 fatty acid biosynthetic pathway means that some or all of the genes in the pathway express active enzymes. It should be understood that “ω-3/ω-6 fatty acid biosynthetic pathway” or “functional ω-3/ω-6 fatty acid biosynthetic pathway” does not imply that all the genes listed in this paragraph are required as a number of fatty acid products will only require the expression of a subset of the genes of this pathway.

The term “desaturase” refers to a polypeptide that can desaturate, i.e., introduce a double bond, in one or more fatty acids to produce a mono- or polyunsaturated fatty acid. Despite use of the omega-reference system throughout the specification in reference to specific fatty acids, it is more convenient to indicate the activity of a desaturase by counting from the carboxyl end of the substrate using the delta-system. Of particular interest herein are Δ12 desaturases that desaturate a fatty acid between the 12^(th) and 13^(th) carbon atoms numbered from the carboxyl-terminal end of the molecule and that catalyze the conversion of oleic acid to LA. Other desaturases relevant to the present disclosure include: Δ15 desaturases that catalyze the conversion of LA to ALA; Δ17 desaturases that desaturate a fatty acid between the 17^(th) and 18^(th) carbon atom numbered from the carboxyl-terminal end of the molecule and which, for example, catalyze the conversion of ARA to EPA and/or DGLA to ETA; Δ6 desaturases that catalyze the conversion of LA to GLA and/or ALA to STA; Δ5 desaturases that catalyze the conversion of DGLA to ARA and/or ETA to EPA; Δ4 desaturases that catalyze the conversion of DPA to DHA; and Δ9 desaturases that catalyze the conversion of palmitate to palmitoleic acid (16:1) and/or stearate to oleic acid (18:1).

The term “elongase” refers to a polypeptide that can elongate a fatty acid carbon chain to produce an acid that is 2 carbons longer than the fatty acid substrate that the elongase acts upon. This process of elongation occurs in a multi-step mechanism in association with fatty acid synthase, whereby CoA is the acyl carrier (Lassner et al., The Plant Cell 8:281-292 (1996)). Briefly, malonyl-CoA is condensed with a long-chain acyl-CoA to yield CO₂ and a β-ketoacyl-CoA (where the acyl moiety has been elongated by two carbon atoms). Subsequent reactions include reduction to β-hydroxyacyl-CoA, dehydration to an enoyl-CoA and a second reduction to yield the elongated acyl-CoA. Examples of reactions catalyzed by elongases are the conversion of GLA to DGLA, STA to ETA and EPA to DPA. Accordingly, elongases can have different specificities (e.g., a C_(16/18) elongase will prefer a C₁₆ substrate, a C_(18/20) elongase will prefer a C₁₈ substrate and a C_(20/22) elongase will prefer a C₂₀ substrate).

The terms “conversion efficiency” and “percent substrate conversion” refer to the efficiency by which a particular enzyme (e.g., a desaturase or elongase) can convert substrate to product. The conversion efficiency is measured according to the following formula: ([product]/[substrate+product])*100, where ‘product’ includes the immediate product and all products in the pathway derived from it.

The term “oleaginous” refers to those organisms that tend to store their energy source in the form of lipid (Weete, In: Fungal Lipid Biochemistry, 2^(nd) ed., Plenum, 1980). Generally, the cellular oil or triacylglycerol content of oleaginous microorganisms follows a sigmoid curve, wherein the concentration of lipid increases until it reaches a maximum at the late logarithmic or early stationary growth phase and then gradually decreases during the late stationary and death phases (Yongmanitchai and Ward, Appl. Environ. Microbiol. 57:419-25 (1991)).

The term “oleaginous yeast” refers to those microorganisms classified as yeasts that can accumulate at least 25% of their dry cell weight as oil. Examples of oleaginous yeast include, but are no means limited to, the following genera: Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces.

The term “fermentable carbon substrate” means a carbon source that a microorganism will metabolize to derive energy. Typical carbon substrates of the invention include, but are not limited to: monosaccharides, oligosaccharides, polysaccharides, alkanes, fatty acids, esters of fatty acids, monoglycerides, carbon dioxide, methanol, formaldehyde, formate and carbon-containing amines.

The term “codon-optimized” as it refers to genes or coding regions of nucleic acid molecules for transformation of various hosts, refers to the alteration of codons in the gene or coding regions of the nucleic acid molecules to reflect the typical codon usage of the host organism without altering the polypeptide encoded by the DNA.

As used herein, an “isolated nucleic acid fragment” is a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. An isolated nucleic acid fragment in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.

A nucleic acid molecule is “hybridizable” to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA molecule, when a single-stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength. Hybridization and washing conditions are well known and exemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2^(nd) ed., Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989), particularly Chapter 11 and Table 11.1 therein (entirely incorporated herein by reference). The conditions of temperature and ionic strength determine the “stringency” of the hybridization. Stringency conditions can be adjusted to screen for moderately similar fragments (such as homologous sequences from distantly related organisms), to highly similar fragments (such as genes that duplicate functional enzymes from closely related organisms). Post-hybridization washes determine stringency conditions. One set of preferred conditions uses a series of washes starting with 6×SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2×SSC, 0.5% SDS at 45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30 min. A more preferred set of stringent conditions uses higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS was increased to 60° C. Another preferred set of highly stringent conditions uses two final washes in 0.1×SSC, 0.1% SDS at 65° C. An additional set of stringent conditions include hybridization at 0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS, for example.

Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of Tm for hybrids of nucleic acids having those sequences. The relative stability (corresponding to higher Tm) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating Tm have been derived (see Sambrook et al., supra, 9.50-9.51). For hybridizations with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al., supra, 11.7-11.8). In one embodiment the length for a hybridizable nucleic acid is at least about 10 nucleotides. Preferably a minimum length for a hybridizable nucleic acid is at least about 15 nucleotides; more preferably at least about 20 nucleotides; and most preferably the length is at least about 30 nucleotides. Furthermore, the skilled artisan will recognize that the temperature and wash solution salt concentration may be adjusted as necessary according to factors such as length of the probe.

A “substantial portion” of an amino acid or nucleotide sequence is that portion comprising enough of the amino acid sequence of a polypeptide or the nucleotide sequence of a gene to putatively identify that polypeptide or gene, either by manual evaluation of the sequence by one skilled in the art, or by computer-automated sequence comparison and identification using algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul, S. F., et al., J. Mol. Biol. 215:403-410 (1993). In general, a sequence of ten or more contiguous amino acids or thirty or more nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene. Moreover, with respect to nucleotide sequences, gene specific oligonucleotide probes comprising 20-30 contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques). In addition, short oligonucleotides of 12-15 bases may be used as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers. Accordingly, a “substantial portion” of a nucleotide sequence comprises enough of the sequence to specifically identify and/or isolate a nucleic acid fragment comprising the sequence. The instant specification teaches the complete amino acid and nucleotide sequence encoding a particular yeast protein. The skilled artisan, having the benefit of the sequences as reported herein, may now use all or a substantial portion of the disclosed sequences for purposes known to those skilled in this art. Accordingly, the instant invention comprises the complete sequences as reported in the accompanying Sequence Listing, as well as substantial portions of those sequences as defined above.

The term “complementary” is used to describe the relationship between nucleotide bases that are capable of hybridizing to one another. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Accordingly, the instant invention also includes isolated nucleic acid fragments that are complementary to the complete sequences as reported in the accompanying Sequence Listing, as well as those substantially similar nucleic acid sequences.

The term “percent identity”, as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. “Identity” and “similarity” can be readily calculated by known methods, including but not limited to those described in: 1.) Computational Molecular Biology (Lesk, A. M., Ed.) Oxford University: NY (1988); 2.) Biocomputing: Informatics and Genome Projects (Smith, D. W., Ed.) Academic: NY (1993); 3.) Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., Eds.) Humana: NJ (1994); 4.) Sequence Analysis in Molecular Biology (von Heinje, G., Ed.) Academic (1987); and 5.) Sequence Analysis Primer (Gribskov, M. and Devereux, J., Eds.) Stockton: NY (1991). Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences is performed using the Clustal method of alignment (Higgins and Sharp, CABIOS. 5:151-153 (1989)) with default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using the Clustal method are: KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

Suitable nucleic acid fragments (isolated polynucleotides of the present invention) encode polypeptides that are at least about 70% identical, preferably at least about 75% identical, and more preferably at least about 80% identical to the amino acid sequence reported herein. Preferred nucleic acid fragments encode amino acid sequences that are about 85% identical to the amino acid sequence reported herein. More preferred nucleic acid fragments encode amino acid sequences that are at least about 90% identical to the amino acid sequence reported herein. Most preferred are nucleic acid fragments that encode amino acid sequences that are at least about 95% identical to the amino acid sequence reported herein. Suitable nucleic acid fragments not only have the above homologies but typically encode a polypeptide having at least 50 amino acids, preferably at least 100 amino acids, more preferably at least 150 amino acids, still more preferably at least 200 amino acids, and most preferably at least 250 amino acids.

“Codon degeneracy” refers to the nature in the genetic code permitting variation of the nucleotide sequence without affecting the amino acid sequence of an encoded polypeptide. The skilled artisan is well aware of the “codon-bias” exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a gene for improved expression in a host cell, it is desirable to design the gene such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.

“Chemically synthesized”, as related to a sequence of DNA, means that the component nucleotides were assembled in vitro. Manual chemical synthesis of DNA may be accomplished using well-established procedures; or automated chemical synthesis can be performed using one of a number of commercially available machines. “Synthetic genes” can be assembled from oligonucleotide building blocks that are chemically synthesized using procedures known to those skilled in the art. These building blocks are ligated and annealed to form gene segments that are then enzymatically assembled to construct the entire gene. Accordingly, the genes can be tailored for optimal gene expression based on optimization of nucleotide sequence to reflect the codon bias of the host cell. The skilled artisan appreciates the likelihood of successful gene expression if codon usage is biased towards those codons favored by the host. Determination of preferred codons can be based on a survey of genes derived from the host cell, where sequence information is available.

“Gene” refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5′ non-coding sequences) and following (3′ non-coding sequences) the coding sequence. “Native gene” refers to a gene as found in nature with its own regulatory sequences. “Chimeric gene” refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. “Endogenous gene” refers to a native gene in its natural location in the genome of an organism. A “foreign” gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes. A “transgene” is a gene that has been introduced into the genome by a transformation procedure. A “codon-optimized gene” is a gene having its frequency of codon usage designed to mimic the frequency of preferred codon usage of the host cell.

“Coding sequence” refers to a DNA sequence that codes for a specific amino acid sequence. “Suitable regulatory sequences” refer to nucleotide sequences located upstream (5′ non-coding sequences), within or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters., translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites and stem-loop structures.

“Promoter” refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3′ to a promoter sequence. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.

The term “3′ non-coding sequences” or “transcription terminator” refers to DNA sequences located downstream of a coding sequence. This includes polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3′ end of the mRNA precursor. The 3′ region can influence the transcription, RNA processing or stability, or translation of the associated coding sequence.

“RNA transcript” refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from post-transcriptional processing of the primary transcript and is referred to as the mature RNA. “Messenger RNA” or “mRNA” refers to the RNA that is without introns and that can be translated into protein by the cell. “cDNA” refers to a double-stranded DNA that is complementary to, and derived from, mRNA. “Sense” RNA refers to RNA transcript that includes the mRNA and so can be translated into protein by the cell. “Antisense RNA” refers to a RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target gene (U.S. Pat. No. 5,107,065; WO 99/28508). The complementarity of an antisense RNA may be with any part of the specific gene transcript, i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, or the coding sequence. “Functional RNA” refers to antisense RNA, ribozyme RNA, or other RNA that is not translated and yet has an effect on cellular processes.

The term “operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.

The term “expression”, as used herein, refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment(s) of the invention. Expression may also refer to translation of mRNA into a polypeptide.

“Mature” protein refers to a post-translationally processed polypeptide; i.e., one from which any pre- or propeptides present in the primary translation product have been removed. “Precursor” protein refers to the primary product of translation of mRNA; i.e., with pre- and propeptides still present. Pre- and propeptides may be (but are not limited to) intracellular localization signals.

“Transformation” refers to the transfer of a nucleic acid molecule into a host organism, resulting in genetically stable inheritance. The nucleic acid molecule may be a plasmid that replicates autonomously, for example; or, it may integrate into the genome of the host organism. Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” or “recombinant” or “transformed” organisms.

The terms “plasmid”, “vector” and “cassette” refer to an extra chromosomal element often carrying genes that are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA fragments. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3′ untranslated sequence into a cell. “Transformation cassette” refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that facilitate transformation of a particular host cell. “Expression cassette” refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that allow for enhanced expression of that gene in a foreign host.

The term “altered biological activity” will refer to an activity, associated with a protein encoded by a nucleotide sequence which can be measured by an assay method, where that activity is either greater than or less than the activity associated with the native sequence. “Enhanced biological activity” refers to an altered activity that is greater than that associated with the native sequence. “Diminished biological activity” is an altered activity that is less than that associated with the native sequence.

The term “homologous recombination” refers to the exchange of DNA fragments between two DNA molecules (during cross over). The fragments that are exchanged are flanked by sites of identical nucleotide sequences between the two DNA molecules (i.e., “regions of homology”). The term “regions of homology” refer to stretches of nucleotide sequence on nucleic acid fragments that participate in homologous recombination that have homology to each other. Effective homologous recombination will take place where these regions of homology are at least about 10 bp in length where at least about 50 bp in length is preferred. Typically fragments that are intended for recombination contain at least two regions of homology where targeted gene disruption or replacement is desired.

The term “sequence analysis software” refers to any computer algorithm or software program that is useful for the analysis of nucleotide or amino acid sequences. “Sequence analysis software” may be commercially available or independently developed. Typical sequence analysis software will include, but is not limited to: 1.) the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wis.); 2.) BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol. 215:403-410 (1990)); 3.) DNASTAR (DNASTAR, Inc. Madison, Wis.); 4.) Sequencher (Gene Codes Corporation, Ann Arbor, Mich.); and 5.) the FASTA program incorporating the Smith-Waterman algorithm (W. R. Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor. Plenum: New York, N.Y.). Within the context of this application it will be understood that where sequence analysis software is used for analysis, that the results of the analysis will be based on the “default values” of the program referenced, unless otherwise specified. As used herein “default values” will mean any set of values or parameters that originally load with the software when first initialized.

Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989) (hereinafter “Maniatis”); by Silhavy, T. J., Bennan, M. L. and Enquist, L. W., Experiments with Gene Fusions, Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1984); and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience (1987).

Microbial Biosynthesis of Fatty Acids

In general, lipid accumulation in oleaginous microorganisms is triggered in response to the overall carbon to nitrogen ratio present in the growth medium (FIG. 1). When cells have exhausted available nitrogen supplies (e.g., when the carbon to nitrogen ratio is greater than about 40), the depletion of cellular adenosine monophosphate (AMP) leads to the cessation of AMP-dependent isocitrate dehydrogenase activity in the mitochondria and the accumulation of citrate, transport of citrate into the cytosol and subsequent cleavage of the citrate by ATP-citrate lyase to yield acetyl-CoA. Acetyl-CoA is the principle building block for de novo biosynthesis of fatty acids. Although any compound that can effectively be metabolized to produce acetyl-CoA can serve as a precursor of fatty acids, glucose is the primary source of carbon in this type of reaction (FIG. 1). Glucose is converted to pyruvate via glycolysis and pyruvate is then transported into the mitochondria where it can be converted to acetyl-CoA by pyruvate dehydrogenase (“PD”). Since acetyl-CoA can not be transported directly across the mitochondrial membrane into the cytoplasm, the two carbons from acetyl-CoA condense with oxaloacetate to yield citrate (catalyzed by citrate synthase). Citrate is transported directly into the cytoplasm, where it is cleaved by ATP-citrate lyase to regenerate acetyl-CoA and oxaloacetate. The oxaloacetate reenters the tricarboxylic acid cycle, via conversion to malate.

The synthesis of malonyl-CoA is the first committed step of fatty acid biosynthesis, which takes place in the cytoplasm. Malonyl-CoA is produced via carboxylation of acetyl-CoA by acetyl-CoA carboxylase (“ACC”). Fatty acid synthesis is catalyzed by a multi-enzyme fatty acid synthase complex (“FAS”) and occurs by the condensation of eight two-carbon fragments (acetyl groups from acetyl-CoA) to form a 16-carbon saturated fatty acid, palmitate. More specifically, FAS catalyzes a series of 7 reactions, which involve the following (Smith, S. FASEB J, 8(15):1248-59 (1994)):

-   -   1. Acetyl-CoA and malonyl-CoA are transferred to the acyl         carrier peptide (ACP) of FAS. The acetyl group is then         transferred to the malonyl group, forming β-ketobutyryl-ACP and         releasing CO₂.     -   2. The β-ketobutyryl-ACP undergoes reduction (via β-ketoacyl         reductase) and dehydration (via β-hydroxyacyl dehydratase) to         form a trans-monounsaturated fatty acyl group.     -   3. The double bond is reduced by NADPH, yielding a saturated         fatty-acyl group two carbons longer than the initial one. The         butyryl-group's ability to condense with a new malonyl group and         repeat the elongation process is then regenerated.     -   4. When the fatty acyl group becomes 16 carbons long, a         thioesterase activity hydrolyses it, releasing free palmitate.

Palmitate (16:0) is the precursor of longer chain saturated and unsaturated fatty acids (e.g., stearic (18:0), palmitoleic (16:1) and oleic (18:1) acids) through the action of elongases and desaturases present in the endoplasmic reticulum membrane. Palmitate and stearate are converted to their unsaturated derivatives, palmitoleic (16:1) and oleic (18:1) acids, respectively, by the action of a Δ9 desaturase.

Triacylglycerols (the primary storage unit for fatty acids) are formed by the esterification of two molecules of acyl-CoA to glycerol-3-phosphate to yield 1,2-diacylglycerol phosphate (commonly identified as phosphatidic acid) (FIG. 1). The phosphate is then removed, by phosphatidic acid phosphatase, to yield 1,2-diacylglycerol. Triacylglycerol is formed upon the addition of a third fatty acid, for example, by the action of a diacylglycerol-acyl transferase.

Biosynthesis of Omega Fatty Acids

Simplistically, the metabolic process that converts LA to GLA, DGLA and ARA (the ω-6 pathway) and ALA to STA, ETA, EPA, DPA and DHA (the ω-3 pathway) involves elongation of the carbon chain through the addition of two-carbon units and desaturation of the molecule through the addition of double bonds (FIG. 2). This requires a series of special desaturation and elongation enzymes present in the endoplasmic reticulum membrane.

ω-6 Fatty Acids

Oleic acid is converted to LA (18:2), the first of the ω-6 fatty acids, by the action of a Δ12 desaturase. Subsequent ω-6 fatty acids are produced as follows: 1.) LA is converted to GLA by the activity of a Δ6 desaturase; 2.) GLA is converted to DGLA by the action of an elongase; and 3.) DGLA is converted to ARA by the action of a Δ5 desaturase.

ω-3 Fatty Acids

Linoleic acid (LA) is converted to ALA, the first of the ω-3 fatty acids, by the action of a Δ15 desaturase. Subsequent ω-3 fatty acids are produced in a series of steps similar to that for the ω-6 fatty acids. Specifically: 1.) ALA is converted to STA by the activity of a Δ6 desaturase; 2.) STA is converted to ETA by the activity of an elongase; and 3.) ETA is converted to EPA by the activity of a Δ5 desaturase. Alternatively, ETA and EPA can be produced from DGLA and ARA, respectively, by the activity of a Δ17 desaturase. EPA can be further converted to DHA by the activity of an elongase and a Δ4 desaturase.

Genes Involved in Omega Fatty Acid Production

Many microorganisms, including algae, bacteria, molds and yeasts, can synthesize PUFAs and omega fatty acids in the ordinary course of cellular metabolism. Particularly well-studied are fungi including Schizochytrium aggregatm, species of the genus Thraustochytrium and Morteriella alpina. Additionally, many dinoflagellates (Dinophyceaae) naturally produce high concentrations of PUFAs. As such, a variety of genes involved in oil production have been identified through genetic means and the DNA sequences of some of these genes are publicly available (non-limiting examples are shown below in Table 2): TABLE 2 Some Publicly Available Genes Involved in PUFA Production Genbank Accession No. Description AY131238 Argania spinosa Δ6 desaturase Y055118 Echium pitardii var. pitardii Δ6 desaturase AY055117 Echium gentianoides Δ6 desaturase AF296076 Mucor rouxii Δ6 desaturase AF007561 Borago officinalis Δ6 desaturase L11421 Synechocystis sp. Δ6 desaturase NM_031344 Rattus norvegicus Δ6 fatty acid desaturase AF465283, Mortierella alpina Δ6 fatty acid desaturase AF465281, AF110510 AF465282 Mortierella isabellina Δ6 fatty acid desaturase AF419296 Pythium irregulare Δ6 fatty acid desaturase AB052086 Mucor circinelloides D6d mRNA for Δ6 fatty acid desaturase AJ250735 Ceratodon purpureus mRNA for Δ6 fatty acid desaturase AF126799 Homo sapiens Δ6 fatty acid desaturase AF126798 Mus musculus Δ6 fatty acid desaturase AF199596, Homo sapiens Δ5 desaturase AF226273 AF320509 Rattus norvegicus liver Δ5 desaturase AB072976 Mus musculus D5D mRNA for Δ5 desaturase AF489588 Thraustochytrium sp. ATCC21685 Δ5 fatty acid desaturase AJ510244 Phytophthora megasperma mRNA for Δ5 fatty acid desaturase AF419297 Pythium irregulare Δ5 fatty acid desaturase AF07879 Caenorhabditis elegans Δ5 fatty acid desaturase AF067654 Mortierella alpina Δ5 fatty acid desaturase AB022097 Dictyostelium discoideum mRNA for Δ5 fatty acid desaturase AF489589.1 Thraustochytrium sp. ATCC21685 Δ4 fatty acid desaturase AX464731 Mortierella alpina elongase gene (also WO 00/12720) AAG36933 Emericella nidulans oleate Δ12 desaturase AF110509 Mortierella alpina Δ12 fatty acid desaturase mRNA AB020033 Mortierella alpina mRNA for Δ12 fatty acid desaturase AAL13300 Mortierella alpina Δ12 fatty acid desaturase AF417244 Mortierella alpina ATCC 16266 Δ12 fatty acid desaturase gene AF161219 Mucor rouxii Δ12 desaturase mRNA X86736 Spiruline platensis Δ12 desaturase AF240777 Caenorhabditis elegans Δ12 desaturase AB007640 Chlamydomonas reinhardtii Δ12 desaturase AB075526 Chlorella vulgaris Δ12 desaturase AP002063 Arabidopsis thaliana microsomal Δ12 desaturase AY332747 Pavlova lutheri Δ4 fatty acid desaturase (des1) mRNA NP_441622, Synechocystis sp. PCC 6803 Δ15 desaturase BAA18302, BAA02924 AAL36934 Perilla frutescens Δ15 desaturase AF338466 Acheta domesticus Δ9 desaturase 3 mRNA AF438199 Picea glauca desaturase Δ9 (Des9) mRNA E11368 Anabaena Δ9 desaturase E11367 Synechocystis Δ9 desaturase D83185 Pichia angusta DNA for Δ9 fatty acid desaturase U90417 Synechococcus vulcanus Δ9 acyl-lipid fatty acid desaturase (desC) gene AF085500 Mortierella alpina Δ9 desaturase mRNA AY504633 Emericella nidulans Δ9 stearic acid desaturase (sdeB) gene NM_069854 Caenorhabditis elegans essential fatty acid desatur- ase, stearoyl-CoA desaturase (39.1 kD) (fat-6) complete mRNA AF230693 Brassica oleracea cultivar Rapid Cycling stearoyl-ACP desaturase (Δ9-BO-1) gene, exon sequence AX464731 Mortierella alpina elongase gene (also WO 02/08401) NM_119617 Arabidopsis thaliana fatty acid elongase 1 (FAE1) (At4g34520) mRNA NM_134255 Mus musculus ELOVL family member 5, elongation of long chain fatty acids (yeast) (Elovl5), mRNA NM_134383 Rattus norvegicus fatty acid elongase 2 (rELO2), mRNA NM_134382 Rattus norvegicus fatty acid elongase 1 (rELO1), mRNA NM_068396, Caenorhabditis elegans fatty acid ELOngation (elo-6), NM_068392, (elo-5), (elo-2), (elo-3), and (elo-9) mRNA NM_070713, NM_068746, NM_064685

Additionally, the patent literature provides many additional DNA sequences of genes (and/or details concerning several of the genes above and their methods of isolation) involved in PUFA production. See, for example: U.S. Pat. No. 5,968,809 (Δ6 desaturases); U.S. Pat. No. 5,972,664 and U.S. Pat. No. 6,075,183 (Δ5 desaturases); WO 91/13972 and U.S. Pat. No. 5,057,419 (Δ9 desaturases); WO 93/11245 (Δ15 desaturases); U.S. 2003/0196217 A1 (Δ17 desaturases), WO 02/090493 (Δ4 desaturases); and WO 00/12720 and U.S. 2002/0139974A1 (elongases). Each of these patents and applications are herein incorporated by reference in their entirety.

Of particular interest herein are Δ12 desaturases, and more specifically, Δ12 desaturases that are suitable for expression in oleaginous yeast (e.g., Yarrowia lipolytica). A variety of sequences encoding fungal Δ12 fatty acid desaturases have been previously disclosed that could be used for heterologous expression in oleaginous Yarrowia lipolytica (e.g., GenBank Accession No's MG36933, AF110509, AAL13300, AF417244, AF161219 (supra)). Additionally, for example, the Δ12 fatty acid desaturases of Glycine max, Brassica napus, Arabidopsis thaliana, Ricinus communis, Zea mays; Neurospora crassa and Botrytis cinerea are disclosed in WO 94/11516, U.S. Pat. No. 5,443,974 and WO 03/099216.

Many factors affect the choice of a specific polypeptide having Δ12 desaturase activity that is to be expressed in a host cell for production of PUFAs (optionally in combination with other desaturases and elongases). Depending upon the host cell, the availability of substrate and the desired end product(s), several polypeptides are of interest; however, considerations for choosing a specific polypeptide having desaturase activity include the substrate specificity of the polypeptide, whether the polypeptide or a component thereof is a rate-limiting enzyme, whether the desaturase is essential for synthesis of a desired polyunsaturated fatty acid and/or co-factors required by the polypeptide. The expressed polypeptide preferably has parameters compatible with the biochemical environment of its location in the host cell. For example, the polypeptide may have to compete for substrate with other enzymes in the host cell. Analyses of the K_(M) and specific activity of the polypeptide are therefore considered in determining the suitability of a given polypeptide for modifying PUFA production in a given host cell. The polypeptide used in a particular host cell is one that can function under the biochemical conditions present in the intended host cell, but otherwise can be any polypeptide having Δ12 desaturase activity capable of modifying the desired fatty acid (i.e., oleic acid). Thus, the sequences may be derived from any source, e.g., isolated from a natural source (from bacteria, algae, fungi, plants, animals, etc.), produced via a semi-synthetic route or synthesized de novo.

Sequence Identification of the Yarrowia lipolytica Δ12 Desaturase

Despite public disclosure of a variety of sequences encoding fungal Δ12 fatty acid desaturases (supra), expression of a native enzyme is preferred over a heterologous (or “foreign”) enzyme since: 1.) the native enzyme is optimized for interaction with other enzymes and proteins within the cell; and 2.) heterologous genes are unlikely to share the same codon preference in the host organism. Additionally, advantages are incurred when the sequence of the native gene is known, as it permits facile disruption of the endogenous gene by targeted disruption.

Concerning disruption of a native Δ12 fatty acid desaturase gene, it may be useful for to engineer an oleaginous yeast that is not capable of producing PUFAs in some embodiments. Commercial applications where this lack of functionality would be desirable include the production of high value cocoa butter substitutes, oxidatively stable oils and specialty fatty acids derived from 18:1 (e.g., hydroxy- and epoxy-fatty acids). Alternatively, oleaginous yeast lacking Δ12 fatty acid desaturase activity could be utilized to produce “pure” ω-3 derivatives of ALA (e.g., STA, ETA, EPA, DPA, DHA) by transforming the organism with the appropriate genes (e.g., Δ6 desaturase, elongase, Δ5 desaturase, Δ4 desaturase) and feeding the organism ALA as a substrate; ω-6 fatty acids would not be synthesized under these conditions (see FIG. 2).

Thus, the Applicants sought to isolate a Δ12 fatty acid desaturase from Yarrowia lipolytica. Comparison of the Δ12 desaturase nucleotide base and deduced amino acid sequences to public databases reveals that the most similar known sequences are about 53% identical to the amino acid sequence of Δ12 desaturase reported herein (SEQ ID NO:24) over a length of 419 amino acids using a Clustal method of alignment (Thompson et. al., Nucleic Acids Res. 22:4673-4680 (1994)). More preferred amino acid fragments are at least about 70%-80% identical to the sequence herein, where those sequences that are 85%-90% identical are particularly suitable and those sequences that are about 95% identical are most preferred. Similarly, preferred Δ12 desaturase encoding nucleic acid sequences corresponding to the instant ORF are those encoding active proteins and which are at least about 70%-80% identical to the nucleic acid sequence of Δ12 desaturase reported herein, where those sequences that are 85%-90% identical are particularly suitable and those sequences that are about 95% identical are most preferred.

Isolation Of Homologs

The Δ12 desaturase nucleic acid fragment of the instant invention may be used to isolate genes encoding homologous proteins from the same or other bacterial, algal, fungal or plant species. Isolation of homologous genes using sequence-dependent protocols is well known in the art. Examples of sequence-dependent protocols include, but are not limited to: 1.) methods of nucleic acid hybridization; 2.) methods of DNA and RNA amplification, as exemplified by various uses of nucleic acid amplification technologies [e.g., polymerase chain reaction (PCR), Mullis et al., U.S. Pat. No. 4,683,202; ligase chain reaction (LCR), Tabor, S. et al., Proc. Acad. Sci. USA 82:1074 (1985); or strand displacement amplification (SDA), Walker, et al., Proc. Natl. Acad. Sci. U.S.A., 89:392 (1992)]; and 3.) methods of library construction and screening by complementation.

For example, genes encoding similar proteins or polypeptides to the desaturase described herein could be isolated directly by using all or a portion of the instant nucleic acid fragments as DNA hybridization probes to screen libraries from any desired yeast or fungus using methodology well known to those skilled in the art (wherein those yeast or fungus producing LA and/or LA-derivatives would be preferred). Specific oligonucleotide probes based upon the instant nucleic acid sequences can be designed and synthesized by methods known in the art (Maniatis, supra). Moreover, the entire sequences can be used directly to synthesize DNA probes by methods known to the skilled artisan (e.g., random primers DNA labeling, nick translation or end-labeling techniques) or RNA probes using available in vitro transcription systems. In addition, specific primers can be designed and used to amplify a part of (or full-length of) the instant sequences. The resulting amplification products can be labeled directly during amplification reactions or labeled after amplification reactions, and used as probes to isolate full-length DNA fragments under conditions of appropriate stringency.

Typically, in PCR-type amplification techniques, the primers have different sequences and are not complementary to each other. Depending on the desired test conditions, the sequences of the primers should be designed to provide for both efficient and faithful replication of the target nucleic acid. Methods of PCR primer design are common and well known in the art (Thein and Wallace, “The use of oligonucleotide as specific hybridization probes in the Diagnosis of Genetic Disorders”, in Human Genetic Diseases: A Practical Approach, K. E. Davis Ed., (1986) pp 33-50, IRL: Herndon, Va.; and Rychlik, W., In Methods in Molecular Biology, White, B. A. Ed., (1993) Vol. 15, pp 31-39, PCR Protocols: Current Methods and Applications. Humania: Totowa, N.J.).

Generally two short segments of the instant sequences may be used in polymerase chain reaction protocols to amplify longer nucleic acid fragments encoding homologous genes from DNA or RNA. The polymerase chain reaction may also be performed on a library of cloned nucleic acid fragments wherein the sequence of one primer is derived from the instant nucleic acid fragments and the sequence of the other primer takes advantage of the presence of the polyadenylic acid tracts to the 3′ end of the mRNA precursor encoding microbial genes.

Alternatively, the second primer sequence may be based upon sequences derived from the cloning vector. For example, the skilled artisan can follow the RACE protocol (Frohman et al., PNAS USA 85:8998 (1988)) to generate cDNAs by using PCR to amplify copies of the region between a single point in the transcript and the 3′ or 5′ end. Primers oriented in the 3′ and 5′ directions can be designed from the instant sequences. Using commercially available 3′ RACE or 5′ RACE systems (BRL, Gaithersburg, Md.), specific 3′ or 5′ cDNA fragments can be isolated (Ohara et al., PNAS USA 86:5673 (1989); Loh et al., Science 243:217 (1989)).

In other embodiments, the instant desaturase sequences may be employed as hybridization reagents for the identification of homologs. The basic components of a nucleic acid hybridization test include a probe, a sample suspected of containing the gene or gene fragment of interest and a specific hybridization method. Probes of the present invention are typically single-stranded nucleic acid sequences that are complementary to the nucleic acid sequences to be detected. Probes are “hybridizable” to the nucleic acid sequence to be detected. The probe length can vary from 5 bases to tens of thousands of bases, and will depend upon the specific test to be done. Typically a probe length of about 15 bases to about 30 bases is suitable. Only part of the probe molecule need be complementary to the nucleic acid sequence to be detected. In addition, the complementarity between the probe and the target sequence need not be perfect. Hybridization does occur between imperfectly complementary molecules with the result that a certain fraction of the bases in the hybridized region are not paired with the proper complementary base.

Hybridization methods are well defined. Typically the probe and sample must be mixed under conditions that will permit nucleic acid hybridization. This involves contacting the probe and sample in the presence of an inorganic or organic salt under the proper concentration and temperature conditions. The probe and sample nucleic acids must be in contact for a long enough time that any possible hybridization between the probe and sample nucleic acid may occur. The concentration of probe or target in the mixture will determine the time necessary for hybridization to occur. The higher the probe or target concentration, the shorter the hybridization incubation time needed. Optionally, a chaotropic agent may be added. The chaotropic agent stabilizes nucleic acids by inhibiting nuclease activity. Furthermore, the chaotropic agent allows sensitive and stringent hybridization of short oligonucleotide probes at room temperature (Van Ness and Chen, Nucl. Acids Res. 19:5143-5151 (1991)). Suitable chaotropic agents include guanidinium chloride, guanidinium thiocyanate, sodium thiocyanate, lithium tetrachloroacetate, sodium perchlorate, rubidium tetrachloroacetate, potassium iodide and cesium trifluoroacetate, among others. Typically, the chaotropic agent will be present at a final concentration of about 3 M. If desired, one can add formamide to the hybridization mixture, typically 30-50% (v/v).

Various hybridization solutions can be employed. Typically, these comprise from about 20 to 60% volume, preferably 30%, of a polar organic solvent. A common hybridization solution employs about 30-50% v/v formamide, about 0.15 to 1 M sodium chloride, about 0.05 to 0.1 M buffers (e.g., sodium citrate, Tris-HCl, PIPES or HEPES (pH range about 6-9)), about 0.05 to 0.2% detergent (e.g., sodium dodecylsulfate), or between 0.5-20 mM EDTA, FICOLL (Pharmacia Inc.) (about 300-500 kdal), polyvinylpyrrolidone (about 250-500 kdal) and serum albumin. Also included in the typical hybridization solution will be unlabeled carrier nucleic acids from about 0.1 to 5 mg/mL, fragmented nucleic DNA (e.g., calf thymus or salmon sperm DNA, or yeast RNA), and optionally from about 0.5 to 2% wt/vol glycine. Other additives may also be included, such as volume exclusion agents that include a variety of polar water-soluble or swellable agents (e.g., polyethylene glycol), anionic polymers (e.g., polyacrylate or polymethylacrylate) and anionic saccharidic polymers (e.g., dextran sulfate).

Nucleic acid hybridization is adaptable to a variety of assay formats. One of the most suitable is the sandwich assay format. The sandwich assay is particularly adaptable to hybridization under non-denaturing conditions. A primary component of a sandwich-type assay is a solid support. The solid support has adsorbed to it or covalently coupled to it immobilized nucleic acid probe that is unlabeled and complementary to one portion of the sequence.

Availability of the instant nucleotide and deduced amino acid sequences facilitates immunological screening of DNA expression libraries. Synthetic peptides representing portions of the instant amino acid sequence may be synthesized. These peptides can be used to immunize animals to produce polyclonal or monoclonal antibodies with specificity for peptides or proteins comprising the amino acid sequences. These antibodies can be then be used to screen DNA expression libraries to isolate full-length DNA clones of interest (Lerner, R. A. Adv. Immunol. 36:1 (1984); Maniatis, supra).

Gene Optimization for Improved Heterologous Expression

A variety of techniques can be utilized to improve the expression of the Δ12 desaturase in an alternate host. Two such techniques include codon-optimization and mutagenesis of the gene.

Codon Optimization

In some embodiments, it may be desirable to modify a portion of the codons encoding the Δ12 desaturase polypeptide, for example, to enhance the expression of the gene encoding that polypeptide in an alternate host (e.g., an oleaginous yeast other than Yarrowia lipolytica).

In general, host preferred codons can be determined within a particular host species of interest by examining codon usage in proteins (preferably those proteins expressed in the largest amount) and determining which codons are used with highest frequency. Then, the coding sequence for the polypeptide of interest having desaturase activity can be synthesized in whole or in part using the codons preferred in the host species. All (or portions) of the DNA also can be synthesized to remove any destabilizing sequences or regions of secondary structure that would be present in the transcribed mRNA. All (or portions) of the DNA also can be synthesized to alter the base composition to one more preferable in the desired host cell.

Mutagenesis

Methods for synthesizing sequences and bringing sequences together are well established in the literature. For example, in vitro mutagenesis and selection, site-directed mutagenesis, error prone PCR (Melnikov et al., Nucleic Acids Research, 27(4):1056-1062 (Feb. 15, 1999)), “gene shuffling” (U.S. Pat. No. 5,605,793; U.S. Pat. No. 5,811,238; U.S. Pat. No. 5,830,721; and U.S. Pat. No. 5,837,458) or other means can be employed to obtain mutations of naturally occurring desaturase genes, such as the Δ12 desaturase described herein. This would permit production of a polypeptide having desaturase activity in vivo with more desirable physical and kinetic parameters for function in the host cell (e.g., a longer half-life or a higher rate of production of a desired PUFA).

If desired, the regions of a desaturase polypeptide important for enzymatic activity can be determined through routine mutagenesis, expression of the resulting mutant polypeptides and determination of their activities. Mutants may include deletions, insertions and point mutations, or combinations thereof. A typical functional analysis begins with deletion mutagenesis to determine the N- and C-terminal limits of the protein necessary for function, and then internal deletions, insertions or point mutants are made to further determine regions necessary for function. Other techniques such as cassette mutagenesis or total synthesis also can be used. Deletion mutagenesis is accomplished, for example, by using exonucleases to sequentially remove the 5′ or 3′ coding regions. Kits are available for such techniques. After deletion, the coding region is completed by ligating oligonucleotides containing start or stop codons to the deleted coding region after the 5′ or 3′ deletion, respectively. Alternatively, oligonucleotides encoding start or stop codons are inserted into the coding region by a variety of methods including site-directed mutagenesis, mutagenic PCR or by ligation onto DNA digested at existing restriction sites. Internal deletions can similarly be made through a variety of methods including the use of existing restriction sites in the DNA, by use of mutagenic primers via site-directed mutagenesis or mutagenic PCR. Insertions are made through methods such as linker-scanning mutagenesis, site-directed mutagenesis or mutagenic PCR. Point mutations are made through techniques such as site-directed mutagenesis or mutagenic PCR.

Chemical mutagenesis also can be used for identifying regions of a desaturase polypeptide important for activity. A mutated construct is expressed, and the ability of the resulting altered protein to function as a desaturase is assayed. Such structure-function analysis can determine which regions may be deleted, which regions tolerate insertions, and which point mutations allow the mutant protein to function in substantially the same way as the native desaturase. All such mutant proteins and nucleotide sequences encoding them that are derived from the desaturase described herein are within the scope of the present invention.

Thus, the present invention comprises the complete sequence of the Δ12 desaturase as reported in the accompanying Sequence Listing, the complement of that complete sequence, substantial portions of that sequence, codon-optimized desaturases derived therefrom and those sequences that are substantially homologous thereto.

Microbial Production of ω-3 and/or ω-6 Fatty Acids

Microbial production of ω-3 and/or ω-6 fatty acids has several advantages over purification from natural sources such as fish or plants. For example:

-   -   1.) Many microbes are known with greatly simplified oil         compositions compared with those of higher organisms, making         purification of desired components easier;     -   2.) Microbial production is not subject to fluctuations caused         by external variables, such as weather and food supply;     -   3.) Microbially produced oil is substantially free of         contamination by environmental pollutants;     -   4.) Microbes can provide PUFAs in particular forms which may         have specific uses; and     -   5.) Microbial oil production can be manipulated by controlling         culture conditions, notably by providing particular substrates         for microbially expressed enzymes, or by addition of compounds         or genetic engineering approaches to suppress undesired         biochemical pathways.         In addition to these advantages, production of ω-3 and/or ω-6         fatty acids from recombinant microbes provides the ability to         alter the naturally occurring microbial fatty acid profile by         providing new biosynthetic pathways in the host or by         suppressing undesired pathways, thereby increasing levels of         desired PUFAs (or conjugated forms thereof) and decreasing         levels of undesired PUFAs (see co-pending U.S. Provisional         Application 60/468,677, herein incorporated entirely by         reference).

Methods for Production of Various ω-3 and/or ω-6 Fatty Acids

It is expected that introduction of chimeric genes encoding the Δ12 desaturase described herein, under the control of appropriate promoters will result in increased production of LA. As such, the present invention encompasses a method for the direct production of PUFAs comprising exposing a fatty acid substrate (i.e., oleic acid) to the PUFA enzyme described herein (i.e., the Δ12 desaturase), such that the substrate is converted to the desired fatty acid product (i.e., LA).

Alternatively, the PUFA gene and its corresponding enzyme product described herein can be used indirectly for the production of PUFAs. Indirect production of PUFAs occurs wherein the fatty acid substrate is converted indirectly into the desired fatty acid product, via means of an intermediate step(s) or pathway intermediate(s). Thus, it is contemplated that the Δ12 desaturase described herein may be expressed in conjunction with one or more genes that encode other enzymes, such that a series of reactions occur to produce a desired product. In a preferred embodiment, for example, a host organism may be co-transformed with a vector comprising additional genes encoding enzymes of the PUFA biosynthetic pathway to result in higher levels of production of ω-3 and/or ω-6 fatty acids (e.g., GLA, DGLA, ARA, ALA, STA, ETA, EPA, DPA and DNA). Specifically, for example, it may be desirable to overexpress the Δ12 desaturase described herein in host cells that are also expressing: 1.) a gene encoding a Δ6 desaturase for the overproduction of GLA; 2.) an expression cassette comprising genes encoding a Δ6 desaturase and a high-affinity elongase for the overproduction of DGLA; 3.) genes encoding a Δ6 desaturase, high-affinity elongase and Δ5 desaturase for the overproduction of ARA; or 4.) genes encoding a Δ6 desaturase, high-affinity elongase, Δ5 desaturase and Δ17 desaturase for the overproduction of EPA. In alternate embodiments, it may be desirable to overexpress the Δ12 desaturase as described herein in cells that are also expressing: 1.) a gene encoding a Δ15 desaturase for the overproduction of ALA; 2.) genes encoding a Δ15 desaturase and Δ6 desaturase for the overproduction of STA; 3.) genes encoding a Δ15 desaturase, Δ16 desaturase and a high-affinity elongase for the overproduction of ETA; or 4.) genes encoding a Δ15 desaturase, Δ6 desaturase, high-affinity elongase and Δ5 desaturase for the overproduction of EPA. As is well known to one skilled in the art, various other combinations of the following enzymatic activities may be useful to express in a host in conjunction with the desaturase herein: a Δ15 desaturase, a Δ4 desaturase, a Δ5 desaturase, a Δ6 desaturase, a Δ17 desaturase, a Δ9 desaturase and/or an elongase (see FIG. 2). The particular genes included within a particular expression cassette will depend on the host cell (and its PUFA profile and/or desaturase profile), the availability of substrate and the desired end product(s).

In alternate embodiments, it may be useful to disrupt a host organism's native Δ12 desaturase, based on the complete sequences described herein, the complement of those complete sequences, substantial portions of those sequences, codon-optimized desaturases derived therefrom and those sequences that are substantially homologous thereto. For example, the targeted disruption of the Δ12 desaturase described herein in Yarrowia lipolytica produces a mutant strain that is unable to synthesize LA. This mutant strain could be useful for: 1.) production of other specialty oils (e.g., high value cocoa butter substitutes, oxidatively stable oils and fatty acids derived from 18:1 such as hydroxy- and epoxy-fatty acids); or 2.) production of “pure” ω-3 fatty acid derivatives of ALA, when the host cells are grown on e.g., ALA (without co-synthesis of ω-6 fatty acids).

Expression Systems, Cassettes and Vectors

The gene and gene product of the instant sequences described herein may be produced in various microbial host cells, particularly in the cells of oleaginous yeasts (e.g., Yarrowia lipolytica). Expression in recombinant microbial hosts may be useful for the production of various PUFA pathway intermediates, or for the modulation of PUFA pathways already existing in the host for the synthesis of new products heretofore not possible using the host.

Microbial expression systems and expression vectors containing regulatory sequences that direct high level expression of foreign proteins are well known to those skilled in the art. Any of these could be used to construct chimeric genes for production of any of the gene products of the instant sequences. These chimeric genes could then be introduced into appropriate microorganisms via transformation to provide high-level expression of the encoded enzymes.

Vectors or DNA cassettes useful for the transformation of suitable host cells are well known in the art. The specific choice of sequences present in the construct is dependent upon the desired expression products (supra), the nature of the host cell and the proposed means of separating transformed cells versus non-transformed cells. Typically, however, the vector or cassette contains sequences directing transcription and translation of the relevant gene(s), a selectable marker and sequences allowing autonomous replication or chromosomal integration. Suitable vectors comprise a region 5′ of the gene that controls transcriptional initiation and a region 3′ of the DNA fragment that controls transcriptional termination. It is most preferred when both control regions are derived from genes from the transformed host cell, although it is to be understood that such control regions need not be derived from the genes native to the specific species chosen as a production host.

Initiation control regions or promoters which are useful to drive expression of the instant ORF in the desired host cell are numerous and familiar to those skilled in the art. Virtually any promoter capable of directing expression of this gene in the selected host cell is suitable for the present invention. Expression in a host cell can be accomplished in a transient or stable fashion. Transient expression can be accomplished by inducing the activity of a regulatable promoter operably linked to the gene of interest. Stable expression can be achieved by the use of a constitutive promoter operably linked to the gene of interest. As an example, when the host cell is yeast, transcriptional and translational regions functional in yeast cells are provided, particularly from the host species. The transcriptional initiation regulatory regions can be obtained, for example, from: 1.) genes in the glycolytic pathway, such as alcohol dehydrogenase, glyceraldehyde-3-phosphate-dehydrogenase (see U.S. Patent Application No. 60/482,263), phosphoglycerate mutase (see U.S. Patent Application No. 60/482,263), fructose-bisphosphate aldolase (see U.S. Patent Application No. 60/519,971), phosphoglucose-isomerase, phosphoglycerate kinase, etc.; or, 2.) regulatable genes such as acid phosphatase, lactase, metallothionein, glucoamylase, the translation elongation factor EF1-α (TEF) protein (U.S. Pat. No. 6,265,185), ribosomal protein S7 (U.S. Pat. No. 6,265,185), etc. Any one of a number of regulatory sequences can be used, depending upon whether constitutive or induced transcription is desired, the efficiency of the promoter in expressing the ORF of interest, the ease of construction and the like.

Nucleotide sequences surrounding the translational initiation codon ‘ATG’ have been found to affect expression in yeast cells. If the instant desaturase is poorly expressed in non-Yarrowia lipolytica yeast, the nucleotide sequences of exogenous genes can be modified to include an efficient yeast translation initiation sequence to obtain optimal gene expression. For expression in yeast, this can be done by site-directed mutagenesis of an inefficiently expressed gene by fusing it in-frame to an endogenous yeast gene, preferably a highly expressed gene. Alternatively, one can determine the consensus translation initiation sequence in the host and engineer this sequence into heterologous genes for their optimal expression in the host of interest.

The termination region can be derived from the 3′ region of the gene from which the initiation region was obtained or from a different gene. A large number of termination regions are known and function satisfactorily in a variety of hosts (when utilized both in the same and different genera and species from where they were derived). The termination region usually is selected more as a matter of convenience rather than because of any particular property. Preferably, the termination region is derived from a yeast gene, particularly Saccharomyces, Schizosaccharomyces, Candida, Yarrowia or Kluyveromyces. The 3′-regions of mammalian genes encoding γ-interferon and α-2 interferon are also known to function in yeast. Termination control regions may also be derived from various genes native to the preferred hosts. Optionally, a termination site may be unnecessary; however, it is most preferred if included.

As one of skill in the art is aware, merely inserting a gene into a cloning vector does not ensure that it will be successfully expressed at the level needed. In response to the need for a high expression rate, many specialized expression vectors have been created by manipulating a number of different genetic elements that control aspects of transcription, translation, protein stability, oxygen limitation, and secretion from the host cell. More specifically, some of the molecular features that have been manipulated to control gene expression include: 1.) the nature of the relevant transcriptional promoter and terminator sequences; 2.) the number of copies of the cloned gene and whether the gene is plasmid-borne or integrated into the genome of the host cell; 3.) the final cellular location of the synthesized foreign protein; 4.) the efficiency of translation in the host organism; 5.) the intrinsic stability of the cloned gene protein within the host cell; and 6.) the codon usage within the cloned gene, such that its frequency approaches the frequency of preferred codon usage of the host cell. Each of these types of modifications are encompassed in the present invention, as means to further optimize expression of the Δ12 desaturase described herein.

Transformation of Microbial Hosts

Once the DNA encoding a polypeptide suitable for expression in an oleaginous yeast has been obtained, it is placed in a plasmid vector capable of autonomous replication in a host cell, or it is directly integrated into the genome of the host cell. Integration of expression cassettes can occur randomly within the host genome or can be targeted through the use of constructs containing regions of homology with the host genome sufficient to target recombination within the host locus. Where constructs are targeted to an endogenous locus, all or some of the transcriptional and translational regulatory regions can be provided by the endogenous locus.

Where two or more genes are expressed from separate replicating vectors, it is desirable that each vector has a different means of selection and should lack homology to the other construct(s) to maintain stable expression and prevent reassortment of elements among constructs. Judicious choice of regulatory regions, selection means and method of propagation of the introduced construct(s) can be experimentally determined so that all introduced genes are expressed at the necessary levels to provide for synthesis of the desired products.

Constructs comprising the gene of interest may be introduced into a host cell by any standard technique. These techniques include transformation (e.g., lithium acetate transformation [Methods in Enzymology, 194:186-187 (1991)]), protoplast fusion, biolistic impact, electroporation, microinjection, or any other method that introduces the gene of interest into the host cell. More specific teachings applicable for oleaginous yeasts (i.e., Yarrowia lipolytica) include U.S. Pat. Nos. 4,880,741 and 5,071,764 and Chen, D. C. et al. (Appl Microbiol Biotechnol. 48(2):232-235 (1997)).

For convenience, a host cell that has been manipulated by any method to take up a DNA sequence (e.g., an expression cassette) will be referred to as “transformed” or “recombinant” herein. The transformed host will have at least one copy of the expression construct and may have two or more, depending upon whether the gene is integrated into the genome, amplified, or is present on an extrachromosomal element having multiple copy numbers. The transformed host cell can be identified by selection for a marker contained on the introduced construct. Alternatively, a separate marker construct may be co-transformed with the desired construct, as many transformation techniques introduce many DNA molecules into host cells. Typically, transformed hosts are selected for their ability to grow on selective media. Selective media may incorporate an antibiotic or lack a factor necessary for growth of the untransformed host, such as a nutrient or growth factor. An introduced marker gene may confer antibiotic resistance, or encode an essential growth factor or enzyme, thereby permitting growth on selective media when expressed in the transformed host. Selection of a transformed host can also occur when the expressed marker protein can be detected, either directly or indirectly. The marker protein may be expressed alone or as a fusion to another protein. The marker protein can be detected by: 1.) its enzymatic activity (e.g., β-galactosidase can convert the substrate X-gal [5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside] to a colored product; luciferase can convert luciferin to a light-emitting product); or 2.) its light-producing or modifying characteristics (e.g., the green fluorescent protein of Aequorea victoria fluoresces when illuminated with blue light). Alternatively, antibodies can be used to detect the marker protein or a molecular tag on, for example, a protein of interest. Cells expressing the marker protein or tag can be selected, for example, visually, or by techniques such as FACS or panning using antibodies. For selection of yeast transformants, any marker that functions in yeast may be used. Desirably, resistance to kanamycin, hygromycin and the amino glycoside G418 are of interest, as well as ability to grow on media lacking uracil or leucine.

Following transformation, substrates suitable for the instant Δ12 desaturase (and optionally other PUFA enzymes that are co-expressed within the host cell) may be produced by the host either naturally or transgenically, or they may be provided exogenously.

Metabolic Engineering of ω-3 and/or ω-6 Fatty Acid Biosynthesis in Microbes

Knowledge of the sequence of the present Δ12 desaturase will be useful for manipulating ω-3 and/or ω-6 fatty acid biosynthesis in oleaginous yeasts, and particularly, in Yarrowia lipolytica. This may require metabolic engineering directly within the PUFA biosynthetic pathway or additional manipulation of pathways that contribute carbon to the PUFA biosynthetic pathway. Methods useful for manipulating biochemical pathways are well known to those skilled in the art.

Techniques to Up-Regulate Desirable Biosynthetic Pathways

Additional copies of desaturase and elongase genes may be introduced into the host to increase the output of the ω-3 and/or ω-6 fatty acid biosynthetic pathways, typically through the use of multicopy plasmids. Expression of the desaturase or elongase genes also can be increased at the transcriptional level through the use of a stronger promoter (either regulated or constitutive) to cause increased expression, by removing/deleting destabilizing sequences from either the mRNA or the encoded protein, or by adding stabilizing sequences to the mRNA (U.S. Pat. No. 4,910,141). Yet another approach to increase expression of heterologous desaturase or elongase genes is to increase the translational efficiency of the encoded mRNAs by replacement of codons in the native gene with those for optimal gene expression in the selected host microorganism.

Techniques to Down-Regulate Undesirable Biosynthetic Pathways

Conversely, biochemical pathways competing with the ω-3 and/or ω-6 fatty acid-biosynthetic pathways for energy or carbon, or native PUFA biosynthetic pathway enzymes that interfere with production of a particular PUFA end-product, may be eliminated by gene disruption or down-regulated by other means (e.g., antisense mRNA). For gene disruption, a foreign DNA fragment (typically a selectable marker gene) is inserted into the structural gene to be disrupted in order to interrupt its coding sequence and thereby functionally inactivate the gene. Transformation of the disruption cassette into the host cell results in replacement of the functional native gene by homologous recombination with the non-functional disrupted gene (see, for example: Hamilton et al. J. Bacteriol. 171:4617-4622 (1989); Balbas et al. Gene 136:211-213 (1993); Gueldener et al. Nucleic Acids Res. 24:2519-2524 (1996); and Smith et al. Methods Mol. Cell. Biol. 5:270-277(1996)).

Antisense technology is another method of down-regulating genes when the sequence of the target gene is known. To accomplish this, a nucleic acid segment from the desired gene is cloned and operably linked to a promoter such that the anti-sense strand of RNA will be transcribed. This construct is then introduced into the host cell and the antisense strand of RNA is produced. Antisense RNA inhibits gene expression by preventing the accumulation of mRNA that encodes the protein of interest. The person skilled in the art will know that special considerations are associated with the use of antisense technologies in order to reduce expression of particular genes. For example, the proper level of expression of antisense genes may require the use of different chimeric genes utilizing different regulatory elements known to the skilled artisan.

Although targeted gene disruption and antisense technology offer effective means of down-regulating genes where the sequence is known, other less specific methodologies have been developed that are not sequence-based. For example, cells may be exposed to UV radiation and then screened for the desired phenotype. Mutagenesis with chemical agents is also effective for generating mutants and commonly used substances include chemicals that affect nonreplicating DNA (e.g., HNO₂ and NH₂OH), as well as agents that affect replicating DNA (e.g., acridine dyes, notable for causing frameshift mutations). Specific methods for creating mutants using radiation or chemical agents are well documented in the art. See, for example: Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, 2^(nd) ed. (1989) Sinauer Associates: Sunderland, Mass.; or Deshpande, Mukund V., Appl. Biochem. Biotechnol., 36:227 (1992).

Another non-specific method of gene disruption is the use of transposable elements or transposons. Transposons are genetic elements that insert randomly into DNA but can be later retrieved on the basis of sequence to determine where the insertion has occurred. Both in vivo and in vitro transposition methods are known. Both methods involve the use of a transposable element in combination with a transposase enzyme. When the transposable element or transposon is contacted with a nucleic acid fragment in the presence of the transposase, the transposable element will randomly insert into the nucleic acid fragment. The technique is useful for random mutagenesis and for gene isolation, since the disrupted gene may be identified on the basis of the sequence of the transposable element. Kits for in vitro transposition are commercially available [see, for example: 1.) The Primer Island Transposition Kit, available from Perkin Elmer Applied Biosystems, Branchburg, N.J., based upon the yeast Ty1 element; 2.) The Genome Priming System, available from New-England Biolabs, Beverly, Mass., based upon the bacterial transposon Tn7; and 3.) the EZ::TN Transposon Insertion Systems, available from Epicentre Technologies, Madison, Wis., based upon the Tn5 bacterial transposable element].

Within the context of the present invention, it may be useful to modulate the expression of the fatty acid biosynthetic pathway by any one of the methods described above. For example, the present invention provides a gene (i.e., a Δ12 desaturase) encoding a key enzyme in the biosynthetic pathway leading to the production of ω-3 and/or ω-6 fatty acids. It will be particularly useful to express this gene in oleaginous yeasts that produce insufficient amounts of 18:2 fatty acids and to modulate the expression of this and other PUFA biosynthetic genes to maximize production of preferred PUFA products using various means for metabolic engineering of the host organism. Likewise, to maximize PUFA production with this gene, it may be necessary to disrupt pathways that compete for the carbon flux directed toward PUFA biosynthesis. In alternate embodiments, it may be desirable to disrupt the Δ12 desaturase herein, to promote synthesis of ω-3 fatty acids while simultaneously preventing co-synthesis of ω-6 fatty acids. In another alternate embodiment it will be possible to regulate the production of ω-3/ω-6 fatty acids by placing the present Δ12 desaturase gene under the control of inducible or regulated promoters.

Preferred Microbial Hosts for Recombinant Expression of Δ12 Desaturase

Host cells for expression of the instant gene and nucleic acid fragments may include microbial hosts that grow on a variety of feedstocks, including simple or complex carbohydrates, organic acids and alcohols, and/or hydrocarbons over a wide range of temperature and pH values. Although the genes described in the instant invention have been isolated for expression in oleaginous yeast, it is contemplated that because transcription, translation and the protein biosynthetic apparatus is highly conserved, any bacteria, yeast, algae and/or filamentous fungus will be a suitable host for expression of the present nucleic acid fragments.

Preferred microbial hosts are oleaginous organisms, such as oleaginous yeasts. These oleaginous organisms are naturally capable of oil synthesis and accumulation, wherein the oil can comprise greater than about 25% of the cellular dry weight, more preferably greater than about 30% of the cellular dry weight, and most preferably greater than about 40% of the cellular dry weight. Genera typically identified as oleaginous yeast include, but are not limited to: Yarrowia, Mortierella, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces. More specifically, illustrative oil-synthesizing yeasts include: Rhodosporidium toruloides, Lipomyces starkeyii, L. lipoferus, Candida revkaufi, C. pulcherrima, C. tropicalis, C. utilis, Trichosporon pullans, T. cutaneum, Rhodotorula glutinus, R. graminis, Mortierella alpina and Yarrowia lipolytica (formerly classified as Candida lipolytica).

Most preferred is the oleaginous yeast Yarrowia lipolytica; and, in a further embodiment, most preferred are the Y. lipolytica strains designated as ATCC #20362, ATCC #8862, ATCC #18944, ATCC #76982 and/or LGAM S(7)1 (Papanikolaou S., and Aggelis G., Bioresour. Technol. 82(1):43-9 (2002)).

Fermentation Processes for PUFA Production

The transformed microbial host cell is grown under conditions that optimize activity of fatty acid biosynthetic genes and produce the greatest and the most economical yield of fatty acids (e.g., LA, which can in turn increase the production of various ω-3 and/or ω-6 fatty acids). In general, media conditions that may be optimized include the type and amount of carbon source, the type and amount of nitrogen source, the carbon-to-nitrogen ratio, the oxygen level, growth temperature, pH, length of the biomass production phase, length of the oil accumulation phase and the time of cell harvest. Microorganisms of interest, such as oleaginous yeast, are grown in complex media (e.g., yeast extract-peptone-dextrose broth (YPD)) or a defined minimal media that lacks a component necessary for growth and thereby forces selection of the desired expression cassettes (e.g., Yeast Nitrogen Base (DIFCO Laboratories, Detroit, Mich.)).

Fermentation media in the present invention must contain a suitable carbon source. Suitable carbon sources may include, but are not limited to: monosaccharides (e.g., glucose, fructose), disaccharides (e.g., lactose, sucrose), oligosaccharides, polysaccharides (e.g., starch, cellulose or mixtures thereof), sugar alcohols (e.g., glycerol) or mixtures from renewable feedstocks (e.g., cheese whey permeate, cornsteep liquor, sugar beet molasses, barley malt). Additionally, carbon sources may include alkanes, fatty acids, esters of fatty acids, monoglycerides, diglycerides, triglycerides, phospholipids and various commercial sources of fatty acids including vegetable oils (e.g., soybean oil) and animal fats. Additionally, the carbon substrate may include one-carbon substrates (e.g., carbon dioxide or methanol) for which metabolic conversion into key biochemical intermediates has been demonstrated. Hence it is contemplated that the source of carbon utilized in the present invention may encompass a wide variety of carbon-containing substrates and will only be limited by the choice of the host organism. Although all of the above mentioned carbon substrates and mixtures thereof are expected to be suitable in the present invention, preferred carbon substrates are sugars and/or fatty acids. Most preferred is glucose and/or fatty acids containing between 10-22 carbons.

Nitrogen may be supplied from an inorganic (e.g., (NH₄)₂SO₄) or organic source (e.g., urea or glutamate). In addition to appropriate carbon and nitrogen sources, the fermentation media must also contain suitable minerals, salts, cofactors, buffers, vitamins, and other components known to those skilled in the art suitable for the growth of the microorganism and promotion of the enzymatic pathways necessary for PUFA production. Particular attention is given to several metal ions (e.g., Mn⁺², Co⁺², Zn⁺², Mg⁺²) that promote synthesis of lipids and PUFAs (Nakahara, T. et al., Ind. Appl. Single Cell Oils, D. J. Kyle and R. Colin, eds. pp 61-97 (1992)).

Preferred growth media in the present invention are common commercially prepared media, such as Yeast Nitrogen Base (DIFCO Laboratories, Detroit, Mich.). Other defined or synthetic growth media may also be used and the appropriate medium for growth of the particular microorganism will be known by one skilled in the art of microbiology or fermentation science. A suitable pH range for the fermentation is typically between about pH 4.0 to pH 8.0, wherein pH 5.5 to pH 7.0 is preferred as the range for the initial growth conditions. The fermentation may be conducted under aerobic or anaerobic conditions, wherein microaerobic conditions are preferred.

Typically, accumulation of high levels of PUFAs in oleaginous yeast cells requires a two-stage process, since the metabolic state must be “balanced” between growth and synthesis/storage of fats. Thus, most preferably, a two-stage fermentation process is necessary for the production of PUFAs in oleaginous yeast. In this approach, the first stage of the fermentation is dedicated to the generation and accumulation of cell mass and is characterized by rapid cell growth and cell division. In the second stage of the fermentation, it is preferable to establish conditions of nitrogen deprivation in the culture to promote high levels of lipid accumulation. The effect of this nitrogen deprivation is to reduce the effective concentration of AMP in the cells, thereby reducing the activity of the NAD-dependent isocitrate dehydrogenase of mitochondria. When this occurs, citric acid will accumulate, thus forming abundant pools of acetyl-CoA in the cytoplasm and priming fatty acid synthesis. Thus, this phase is characterized by the cessation of cell division followed by the synthesis of fatty acids and accumulation of oil.

Although cells are typically grown at about 30° C., some studies have shown increased synthesis of unsaturated fatty acids at lower temperatures (Yongmanitchai and Ward, Appl. Environ. Microbiol. 57:419-25 (1991)). Based on process economics, this temperature shift should likely occur after the first phase of the two-stage fermentation, when the bulk of the organisms' growth has occurred.

It is contemplated that a variety of fermentation process designs may be applied, where commercial production of omega fatty acids using the instant Δ12 desaturase is desired. For example, commercial production of PUFAs from a recombinant microbial host may be produced by a batch, fed-batch or continuous fermentation process.

A batch fermentation process is a closed system wherein the media-composition is set at the beginning of the process and not subject to further additions beyond those required for maintenance of pH and oxygen level during the process. Thus, at the beginning of the culturing process the media is inoculated with the desired organism and growth or metabolic activity is permitted to occur without adding additional substrates (i.e., carbon and nitrogen sources) to the medium. In batch processes the metabolite and biomass compositions of the system change constantly up to the time the culture is terminated. In a typical batch process, cells proceed through a static lag phase to a high-growth log phase and finally to a stationary phase, wherein the growth rate is diminished or halted. Left untreated, cells in the stationary phase will eventually die. A variation of the standard batch process is the fed-batch process, wherein the substrate is continually added to the fermentor over the course of the fermentation process. A fed-batch process is also suitable in the present invention. Fed-batch processes are useful when catabolite repression is apt to inhibit the metabolism of the cells or where it is desirable to have limited amounts of substrate in the media at any one time. Measurement of the substrate concentration in fed-batch systems is difficult and therefore may be estimated on the basis of the changes of measurable factors such as pH, dissolved oxygen and the partial pressure of waste gases (e.g., CO₂). Batch and fed-batch culturing methods are common and well known in the art and examples may be found in Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, 2^(nd) ed., (1989) Sinauer Associates: Sunderland, Mass.; or Deshpande, Mukund V., Appl. Biochem. Biotechnol., 36:227 (1992), herein incorporated by reference.

Commercial production of omega fatty acids using the instant Δ12 desaturase may also be accomplished by a continuous fermentation process wherein a defined media is continuously added to a bioreactor while an equal amount of culture volume is removed simultaneously for product recovery. Continuous cultures generally maintain the cells in the log phase of growth at a constant cell density. Continuous or semi-continuous culture methods permit the modulation of one factor or any number of factors that affect cell growth or end product concentration. For example, one approach may limit the carbon source and allow all other parameters to moderate metabolism. In other systems, a number of factors affecting growth may be altered continuously while the cell concentration, measured by media turbidity, is kept constant. Continuous systems strive to maintain steady state growth and thus the cell growth rate must be balanced against cell loss due to media being drawn off the culture. Methods of modulating nutrients and growth factors for continuous culture processes, as well as techniques for maximizing the rate of product formation, are well known in the art of industrial microbiology and a variety of methods are detailed by Brock, supra.

Purification of PUFAs

The PUFAs may be found in the host microorganism as free fatty acids or in esterified forms such as acylglycerols, phospholipids, sulfolipids or glycolipids, and may be extracted from the host cell through a variety of means well-known in the art. One review of extraction techniques, quality analysis and acceptability standards for yeast lipids is that of Z. Jacobs (Critical Reviews in Biotechnology 12(5/6):463-491 (1992)). A brief review of downstream processing is also available by A. Singh and O. Ward (Adv. Appl. Microbiol. 45:271-312 (1997)).

In general, means for the purification of PUFAs may include extraction with organic solvents, sonication, supercritical fluid extraction (e.g., using carbon dioxide), saponification and physical means such as presses, or combinations thereof. Of particular interest is extraction with methanol and chloroform in the presence of water (E. G. Bligh & W. J. Dyer, Can. J. Biochem. Physiol. 37:911-917 (1959)). Where desirable, the aqueous layer can be acidified to protonate negatively-charged moieties and thereby increase partitioning of desired products into the organic layer. After extraction, the organic solvents can be removed by evaporation under a stream of nitrogen. When isolated in conjugated forms, the products may be enzymatically or chemically cleaved to release the free fatty acid or a less complex conjugate of interest, and can then be subject to further manipulations to produce a desired end product. Desirably, conjugated forms of fatty acids are cleaved with potassium hydroxide.

If further purification is necessary, standard methods can be employed. Such methods may include extraction, treatment with urea, fractional crystallization, HPLC, fractional distillation, silica gel chromatography, high-speed centrifugation or distillation, or combinations of these techniques. Protection of reactive groups, such as the acid or alkenyl groups, may be done at any step through known techniques (e.g., alkylation, iodination). Methods used include methylation of the fatty acids to produce methyl esters. Similarly, protecting groups may be removed at any step. Desirably, purification of fractions containing GLA, STA, ARA, DHA and EPA may be accomplished by treatment with urea and/or fractional distillation.

DESCRIPTION OF PREFERRED EMBODIMENTS

The ultimate goal of the work described herein is the development of an oleaginous yeast that accumulates oils enriched in ω-3 and/or ω-6 PUFAs. Toward this end, Δ12 desaturases must be identified that function efficiently in oleaginous yeasts, to enable synthesis and high accumulation of preferred PUFAs in these hosts. Identification of efficient Δ12 desaturases is also necessary to manipulate the ratio of ω-3 to ω-6 PUFAs produced in host cells.

In the present invention, Applicants have isolated and cloned the only gene in Yarrowia lipolytica that encodes a Δ12 desaturase enzyme. Confirmation of this gene's activity was provided based upon: 1.) the lack of detectable LA in a strain wherein disruption of the native Δ12 desaturase by targeted gene replacement through homologous recombination had occurred (Example 2); 2.) restoration of LA biosynthesis (complementation) in the disrupted strain upon transformation with the chimeric gene (Example 4); and 3.) the overproduction of LA in wild type cells upon transformation with the chimeric gene (Example 4). Thus, this Δ12 desaturase gene is useful for expression in various microbial hosts, and particularly for overexpression in oleaginous yeasts (e.g., the native host Yarrowia lipolytica). Additional benefits may result since expression of the Δ12 desaturase can also be put under the control of strong constitutive or regulated promoters that do not have the regulatory constraints of the native gene.

Following the initial demonstration of functionality of the Δ12 desaturase in Yarrowia lipolytica, the Applicants then explored methods of optimizing PUFA production within this model host organism. Specifically, a Δ12 desaturase-disrupted host strain of Y. lipolytica was created and transformed with an expression cassette comprising a heterologous Δ6 desaturase, elongase, Δ5 desaturase and Δ17 desaturase. When fed ALA as a substrate, the transformed host was able to produce STA without co-synthesis of any ω-6 fatty acid (Example 8). Thus, this work demonstrated that upon transformation with appropriate genes of the ω-3 biosynthetic pathway and feeding of ALA as a substrate, only ω-3 fatty acids (e.g., ETA, EPA, DPA, DHA) could be synthesized (i.e., without co-synthesis of ω-6 fatty acids) in Yarrowia strains lacking Δ12 desaturase activity.

EXAMPLES

The present invention is further defined in the following Examples. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

General Methods

Standard recombinant DNA and molecular cloning techniques used in the Examples are well known in the art and are described by: 1.) Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989) (Maniatis); 2.) T. J. Silhavy, M. L. Bennan, and L. W. Enquist, Experiments with Gene Fusions; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1984); and 3.) Ausubel, F. M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience (1987).

Materials and methods suitable for the maintenance and growth of microbial cultures are well known in the art. Techniques suitable for use in the following Examples may be found as set out in Manual of Methods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. Briggs Phillips, Eds), American Society for Microbiology: Washington, D.C. (1994)); or by Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, 2^(nd) ed., Sinauer Associates: Sunderland, Mass. (1989). All reagents, restriction enzymes and materials used for the growth and maintenance of microbial cells were obtained from Aldrich Chemicals (Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.), GIBCO/BRL (Gaithersburg, Md.), or Sigma Chemical Company (St. Louis, Mo.), unless otherwise specified.

E. coli TOP10 cells and E. coli Electromax DH10B cells were obtained from Invitrogen (Carlsbad, Calif.). Max Efficiency competent cells of E. coli DH5α were obtained from GIBCO/BRL (Gaithersburg, Md.). E. coli (XL1-Blue) competent cells were purchased from the Stratagene Company (San Diego, Calif.). E. coli strains were typically grown at 37° C. on Luria Bertani (LB) plates.

General molecular cloning was performed according to standard methods (Sambrook et al., supra). Oligonucleotides were synthesized by Sigma-Genosys (Spring, Tex.). PCR products were cloned into Promega's pGEM-T-easy vector (Madison, Wis.).

DNA sequence was generated on an ABI Automatic sequencer using dye terminator technology (U.S. Pat. No. 5,366,860; EP 272,007) using a combination of vector and insert-specific primers. Sequence editing was performed in Sequencher (Gene Codes Corporation, Ann Arbor, Mich.). All sequences represent coverage at least two times in both directions. Comparisons of genetic sequences were accomplished using DNASTAR software (DNA Star, Inc.).

The meaning of abbreviations is as follows: “sec” means second(s), “min” means minute(s), “h” means hour(s), “d” means day(s), “μL” means microliter(s), “mL” means milliliter(s), “L” means liter(s), “μM” means micromolar, “mM” means millimolar, “M” means molar, “mmol” means millimole(s), “μmole” mean micromole(s), “g” means gram(s), “μg” means microgram(s), “ng” means nanogram(s), “U” means unit(s), “bp” means base pair(s) and “kB” means kilobase(s).

Cultivation of Yarrowia lipolytica

Yarrowia lipolytica strains ATCC #76982 and ATCC #90812 were purchased from the American Type Culture Collection (Rockville, Md.). Y. lipolytica strains were usually grown at 28° C. on YPD agar (1% yeast extract, 2% bactopeptone, 2% glucose, 2% agar). For selection of transformants, minimal medium (0.17% yeast nitrogen base (DIFCO Laboratories, Detroit, Mich.) without ammonium sulfate or amino acids, 2% glucose, 0.1% proline, pH 6.1) was used. Supplements of adenine, leucine, lysine and/or uracil were added as appropriate to a final concentration of 0.01%.

Fatty Acid Analysis of Yarrowia lipolytica

For fatty acid analysis, cells were collected by centrifugation and lipids were extracted as described in Bligh, E. G. & Dyer, W. J. (Can. J. Biochem. Physiol. 37:911-917 (1959)). Fatty acid methyl esters were prepared by transesterification of the lipid extract with sodium methoxide (Roughan, G., and Nishida I. Arch Biochem Biophys. 276(1):38-46 (1990)) and subsequently analyzed with a Hewlett-Packard 6890 GC fitted with a 30-m×0.25 mm (i.d.) HP-INNOWAX (Hewlett-Packard) column. The oven temperature was from 170° C. (25 min hold) to 185° C. at 3.5° C./min.

For direct base transesterification, Yarrowia culture (3 mL) was harvested, washed once in distilled water, and dried under vacuum in a Speed-Vac for 5-10 min. Sodium methoxide (100 μl of 1%) was added to the sample, and then the sample was vortexed and rocked for 20 min. After adding 3 drops of 1 M NaCl and 400 μl hexane, the sample was vortexed and spun. The upper layer was removed and analyzed by GC as described above.

Example 1 Construction of Plasmids Suitable for Gene Expression in Yarrowia lipolytica

The present Example describes the construction of plasmids pY5, pY5-4, pY5-13 and pY5-20.

Construction of Plasmid pY5

The plasmid pY5, a derivative of pINA532 (a gift from Dr. Claude Gaillardin, Insitut National Agronomics, Centre de biotechnologie Agro-Industrielle, laboratoire de Genetique Moleculaire et Cellularie INRA-CNRS, F-78850 Thiverval-Grignon, France), was constructed for expression of heterologous genes in Yarrowia lipolytica, as diagrammed in FIG. 3.

First, the partially-digested 3598 bp EcoRI fragment containing the ARS18 sequence and LEU2 gene of pINA532 was subcloned into the EcoRI site of pBluescript (Strategene, San Diego, Calif.) to generate pY2. The TEF promoter (Muller S., et al. Yeast, 14: 1267-1283 (1998)) was amplified from Yarrowia lipolytica genomic DNA by PCR using TEF5′ (SEQ ID NO:1) and TEF3′ (SEQ ID NO:2) as primers. PCR amplification was carried out in a 50 μl total volume containing: 100 ng Yarrowia genomic DNA, PCR buffer containing 10 mM KCl, 10 mM (NH₄)₂SO₄, 20 mM Tris-HCl (pH 8.75), 2 mM MgSO₄, 0.1% Triton X-100, 100 μg/mL BSA (final concentration), 200 μM each deoxyribonucleotide triphosphate, 10 pmole of each primer and 1 μl of PfuTurbo DNA polymerase (Stratagene). Amplification was carried out as follows: initial denaturation at 95° C. for 3 min, followed by 35 cycles of the following: 95° C. for 1 min, 56° C. for 30 sec, 72° C. for 1 min. A final extension cycle of 72° C. for 10 min was carried out, followed by reaction termination at 4° C. The 418 bp PCR product was ligated into pCR-Blunt to generate pIP-tef. The BamHI/EcoRV fragment of pIP-tef was subcloned into the BamHI/SmaI sites of pY2 to generate pY4.

The XPR2 transcriptional terminator was amplified by PCR using pINA532 as template and XPR5′ (SEQ ID NO:3) and XPR3′ (SEQ ID NO:4) as primers. The PCR amplification was carried out in a 50 μl total volume, using the components and conditions described above. The 179 bp PCR product was digested with SacII and then ligated into the SacII site of pY4 to generate pY5. Thus, pY5 (shown in FIGS. 3 and 4) is useful as a Yarrowia-E. coli shuttle plasmid containing:

-   -   1.) a Yarrowia autonomous replication sequence (ARS18);     -   2.) a ColE1 plasmid origin of replication;     -   3.) an ampicillin-resistance gene (Amp^(R)), for selection in E.         coli;     -   4.) a Yarrowia LEU2 gene (E.C. 1.1.1.85, encoding         isopropylmalate isomerase), for selection in Yarrowia;     -   5.) the translation elongation promoter (TEF P), for expression         of heterologous genes in Yarrowia; and     -   6.) the extracellular protease gene terminator (XPR2) for         transcriptional termination of heterologous gene expression in         Yarrowia.         Construction of Plasmids pY-4, pY5-13 And pY5-20

pY5-4 and pY5-13 (FIG. 4) were constructed as derivatives of pY5 to faciliate subcloning and heterologous gene expression in Yarrowia lipolytica.

Specifically, pY5-4 was constructed by three rounds of site-directed mutagenesis using pY5 as template. A NcoI site located inside the Leu2 reporter gene was eliminated from pY5 using oligonucleotides YL1 and YL2 (SEQ ID NOs:5 and 6) to generate pY5-1. A NcoI site was introduced into pY5-1 between the TEF promoter and XPR2 transcriptional terminator by site-directed mutagenesis using oligonucleotides YL3 and YL4 (SEQ ID NOs:7 and 8) to generate pY5-2. A PacI site was then introduced into pY5-2 between the TEF promoter and XPR2 transcriptional terminator using oligonucleotides YL23 and YL24 (SEQ ID NOs:9 and 10) to generate pY5-4.

pY5-13 was constructed by 6 rounds of site-directed mutagenesis using pY5 as template. Both SalI and ClaI sites were eliminated from pY5 by site-directed mutagenesis using oligonucleotides YL5 and YL6 (SEQ ID NOs:11 and 12) to generate pY5-5. A SalI site was introduced into pY5-5 between the Leu2 gene and the TEF promoter by site-directed mutagenesis using oligonucleotides YL9 and YL10 (SEQ ID NOs:13 and 14) to generate pY5-6. A PacI site was introduced into pY5-6 between the LEU2 gene and ARS18 using oligonucleotides YL7 and YL8 (SEQ ID NOs:15 and 16) to generate pY5-8. A NcoI site was introduced into pY5-8 around the translation start codon of the TEF promoter using oligonucleotides YL3 and YL4 (SEQ ID NOs:7 and 8) to generate pY5-9. The NcoI site inside the Leu2 gene of pY5-9 was eliminated using YL1 and YL2 oligonucleotides (SEQ ID NOs:5 and 6) to generate pY5-12. Finally, a BsiWI site was introduced into pY5-12 between the ColEI and XPR2 region using oligonucleotides YL61 and YL62 (SEQ ID NOs:17 and 18) to generate pY5-13.

Plasmid pY20 is a derivative of pY5. It was constructed by inserting a Not I fragment containing a chimeric hygromycin resistance gene (hygromycin-B phosphotransferase; GenBank Accession No. P00557) into the Not/site of pY5. The chimeric gene had the hygromycin resistance ORF under the control of a Yarrowia lipolytica TEF promoter.

Example 2 Cloning of the Partial Yarrowia lipolytica Δ12 Desaturase and Disruption of the Endogenous Δ12 Desaturase Gene

Based on the fatty acid composition of wildtype Yarrowia lipolytica (ATCC #76982) which demonstrated that the organism could make LA (18:2) but not ALA (18:3), it was assumed that Y. lipolytica would likely contain gene(s) having Δ12 desaturase activity but not Δ15 desaturase activity. Thus, the present Example describes the use of degenerate PCR primers to isolate a partial coding sequence of the Y. lipolytica Δ12 desaturase and the use of the partial sequence to disrupt the native gene.

Cloning of the Partial Putative Δ12 Desaturase Sequence from Y. lipolytica by PCR Using Degenerate PCR Primers

Genomic DNA was isolated from Y. lipolytica (ATCC #76982) using DNeasy Tissue Kit (Qiagen, Catalog #69504) and resuspended in kit buffer AE at a DNA concentration of 0.5 μg/μl. PCR amplifications were performed using the genomic DNA as template and several sets of degenerate primers made to amino acid sequences conserved between different fungal Δ12 desaturases (i.e., Mortierella alpina, Mucor rouxii, Emericella nidulans and Pichia augusta). The best results were obtained with a set of upper and lower degenerate primers, P73 and P76, respectively, as shown in the Table below. TABLE 3 Degenerate Primers Used For Amplification Of The Partial Putative Δ12 Desaturase Degenerate Corresponding Primer Nucleotide Amino Acid Set Description Sequence Sequence P73 (32) 26- 5′- WVLGHECGH mers TGGGTCCTGGGCCAYG (SEQ ID NO:20) ARTGYGGNCA-3′ (SEQ ID NO:19) P76 (64) 30- 5′- (M/I)PFYHAEEAT mers GGTGGCCTCCTCGGCG (SEQ ID NO:22) TGRTARAANGGNAT- 3′ (SEQ ID NO:21) [Note: Abbreviations are standard for nudeotides and proteins. The-nucleic acid degeneracy code used is as follows: R = A/G; Y = C/T; and N = A/C/G/T.]

The PCR was carried out in an Eppendorf Mastercycler Gradient thermocycler according to the manufacturer's recommendations. Amplification was carried out as follows: initial denaturation at 95° C. for 1 min, followed by 30 cycles of denaturation at 95° C. for 30 sec, annealing at 58° C. for 1 min, and elongation at 72° C. for 1 min. A final elongation cycle at 72° C. for 10 min was carried out, followed by reaction termination at 4° C.

The expected (ca. 740 bp) size PCR product was detected by agarose gel electrophoresis, isolated, purified, cloned into a pTA vector (Invitrogen) and sequenced. The resultant sequence (contained within SEQ ID NO:23) had homology to known Δ12 desaturases, based on BLAST program analysis (Basic Local Alignment Search Tool; Altschul, S. F., et al., J. Mol. Biol. 215:403-410 (1993).

Targeted Disruption of the Yarrowia lipolytica Δ12 Desaturase Gene

Targeted disruption of the native Δ12 desaturase gene in Y. lipolytica #76982 was carried out by homologous recombination-mediated replacement of the Δ12 desaturase gene with a targeting cassette designated as pY23D12. pY23D12 was derived from plasmid pY20 (Example 1). Specifically, pY23D12 was created by inserting a 642 bp Hind III/Eco RI fragment into similarly linearized pY20. This 642 bp fragment consisted of (in 5′ to 3′ orientation): 3′ homologous sequence from position +718 to +1031 (of the coding sequence (ORF) in SEQ ID NO:23), a Bgl II restriction site and 5′ homologous sequence from position +403 to +717 (of the coding sequence (ORF) in SEQ ID NO:23). The fragment was prepared by PCR amplification of 3′ and 5′ sequences from the 642 bp PCR product using sets of PCR primers P99 and P100 (SEQ ID NOs:25 and 26) and P101 and P102 (SEQ ID NOs:27 and 28), respectively.

pY23D12 was linearized by Bgl II restriction digestion and transformed into mid-log phase Y. lipolytica cells by the lithium acetate method according to the method of Chen, D. C. et al. (Appl Microbiol Biotechnol. 48(2):232-235 (1997)). Briefly, Y. lipolytica ATCC #76982 was streaked onto a YPD plate and grown at 30° C. for approximately 18 hr. Several large loopfuls of cells were scraped from the plate and resuspended in 1 mL of transformation buffer containing:

-   -   2.25 mL of 50% PEG, average MW 3350;     -   0.125 mL of 2 M Li acetate, pH 6.0;     -   0.125 mL of 2 M DTT; and,     -   50 μg sheared salmon sperm DNA.

About 500 ng of plasmid DNA were incubated in 100 μl of resuspended cells, and maintained at 39° C. for 1 hr with vortex mixing at 15 min intervals. The cells were plated onto YPD hygromycin selection plates and maintained at 30° C. for 2 to 3 days.

Four hygromycin-resistant colonies were isolated and screened for targeted disruption by PCR. One set of PCR primers (P119 [SEQ ID NO:29] and P120 [SEQ ID NO:30]) was designed to amplify a specific junction fragment following homologous recombination. Another set of PCR primers (P121 [SEQ ID NO:31] and P122 [SEQ ID NO:32]) was designed to detect the native gene. Three of the four hygromycin-resistant colonies were positive for the junction fragment and negative for the native fragment, thus confirming targeted integration.

Determination of Fatty Acid Profile in the Δ12 Desaturase-Disrupted Strain

Disruption of the Δ12 desaturase gene was further confirmed by GC analysis of the total lipids in one of the disrupted strains, designated as “Q-d12D”. Single colonies of wild type (ATCC #76982) and Q-d12D Y. lipolytica were each grown in 3 mL minimal media (formulation/L: 20 g glucose, 1.7 g yeast nitrogen base, 1 g L-proline, 0.1 g L-adenine, 0.1 g L-lysine, pH 6.1) at 30° C. to an OD₆₀₀˜1.0. The cells were harvested, washed in distilled water, speed vacuum dried and subjected to direct trans-esterification and GC analysis (as described in the General Methods).

The fatty acid profile of wildtype Yarrowia and the transformant Q-d12D comprising the disrupted Δ12 desaturase are shown below in Table 4. Fatty acids are identified as 16:0 (palmitate), 16:1 (palmitoleic acid), 18:0, 18:1 (oleic acid) and 18:2 (LA); and the composition of each is presented as a % of the total fatty acids. TABLE 4 Fatty Acid Composition (% Of Total Fatty Acids) In Wildtype And Transformant Yarrowia lipolytica Strain 16:0 16:1 18:0 18:1 18:2 Wild type 11 14 2 33 34 Q-d12D disrupted 6 15 1 74 nd *nd = not detectable Results indicated that the native Δ12 desaturase gene in the Q-d12D strain was inactivated. Thus, it was possible to conclude that there was only one gene encoding a functional Δ12 desaturase in Yarrowia lipolytica ATCC #76982.

Example 3 Cloning of the Full-Length Yarrowia lipolytica Δ12 Desaturase Gene

The present Example describes the recovery of the genomic sequences flanking the disrupted gene by plasmid rescue, using the sequence in the rescued plasmid to PCR the intact open reading frame of the native gene. The full-length gene and its deduced amino acid sequence is compared to other fungal desaturases.

Plasmid Rescue of the Yarrowia lipolytica Δ12 Desaturase Gene

Since the Δ12 desaturase gene was disrupted by the insertion of the entire pY23D12 vector that also contained an E. coli ampicillin-resistant gene and E. coli ori, it was possible to rescue the flanking sequences in E. coli. For this, genomic DNA of Y. lipolytica strain Q-d12D (carrying the disrupted Δ12 desaturase gene; Example 2) was isolated using the DNeasy Tissue Kit. Then, 10 μg of the genomic DNA was digested with 50 μl of restriction enzymes Age I, Avr II, Nhe I and Sph I in a reaction volume of 200 μl. Digested DNA was extracted with phenol:chloroform and resuspended in 40 μl deionized water. The digested DNA (10 μl) was self-ligated in a 200 μl ligation mixture containing 3 U T4 DNA ligase. Ligation was carried out at 16° C. for 12 hrs. The ligated DNA was extracted with phenol:chloroform and resuspended in 40 μl deionized water. Finally, 1 μl of the resuspended ligated DNA was used to transform E. coli by electroporation and plated onto LB plates containing ampicillin (Ap). Ap-resistant colonies were isolated and analyzed for the presence of plasmids by miniprep. The following insert sizes were found in the recovered or rescued plasmids (Table 5): TABLE 5 Insert Sizes Of Recovered Plasmids, According To Restriction Enzyme Enzyme Plasmid Insert Size (kB) AgeI 1.6 AvrII 2.5 NheI 9.4 SphI 6.6 Sequencing of the plasmids was initiated with sequencing primers P99 (SEQ ID NO:25) and P102 (SEQ ID NO:28).

Based on the sequencing results, a full-length gene encoding the Yarrowia lipolytica Δ12 desaturase gene was assembled (1-936 bp; SEQ ID NO:23). Specifically, SEQ ID NO:23 encoded an open reading frame of 1257 bases (nucleotides +283 to +1539), while the deduced amino acid sequence was 419 residues in length (SEQ ID NO:24).

The Yarrowia lipolytica Δ12 desaturase protein (SEQ ID NO:24) was used as a query against available sequence databases of filamentous fungi, including: 1.) public databases of Neurospora crassa, Magnaporthe grisea, Aspergillus nidulans and Kluyveromuces lactis; and 2.) a DuPont EST library of Fusarium moniliforme strain M-8114 (E.I. du Pont de Nemours and Co., Inc., Wilmington, Del.) (F. moniliforme strain M-8114 available from the Fusarium Research Center, University Park, Pa.; see also Plant Disease 81(2): 211-216. (1997)). These BLAST searches identified the following homologs (Table 6). TABLE 6 Description of Δ12 Desaturase Homologs Source Symbol Organism Contig 1.122 (scaffold 9) in the A. nidulans An1 Aspergillus genome project (sponsored by the Center nidulans for Genome Research (CGR), Cambridge, MA. Contig 1.15 (scaffold 1) in the A. nidulans An2 Aspergillus genome project; AAG36933 nidulans DuPont EST sequence database, U.S. Fm1 Fusarium Provisional Application No. 60/519191 moniliforme DuPont EST sequence database, U.S. Fm2 Fusarium Provisional Application No. 60/519191 moniliforme Ctg4369-0000002-2.1 in the Genolevures Kl Kluyveromyces project. lactis Locus MG08474.1 in contig 2.1597 in the M. Mg1 Magnaporthe grisea genome project (sponsored by the grisea CGR and International Rice Blast Genome Consortium. Locus MG01985.1 in contig 2.375 in the M. Mg2 Magnaporthe grisea genome project grisea GenBank Accession No. AABX01000374 Nc1 Neurospora crassa GenBank Accession No. AABX01000577 Nc2 Neurospora crassa All of the homologs were either unannotated or annotated as a fatty acid desaturase. Furthermore, the nucleotide sequences from A. nidulans were incomplete and/or genomic with putative intron sequences; the Applicants made a tentative assembly of the deduced amino acids for comparison with amino acid sequences from the other homologs.

A comparison of the deduced amino acid sequence of the Yarrowia lipolytica Δ12 desaturase (SEQ ID NO:24) was made with the fungal homologs shown above in Table 6 and other known Δ12 desaturases, as described below in Table 7. TABLE 7 Known Δ12 Desaturases Source Symbol Organism GenBank Accession No. AAG36933 En Emericella nidulans GenBank Accession No. AF110509 Ma Mortierella alpina GenBank Accession No. AB020033 MaB Mortierella alpina GenBank Accession No. AAL13300; MaC Mortierella alpina AF417244 GenBank Accession No. AF161219 Mr Mucor rouxii Ctg1334-0000001-1.1. Pa Pichia augusta (see Genolevures project.)

Specifically, the analysis was performed using the ClustalW alignment algorithm (Slow/Accurate, Gonnet option; Thompson et. al., Nucleic Acids Res. 22:4673-4680 (1994)) of the DNASTAR software package (DNASTAR Inc., Madison, Wis.). This comparison revealed the Pair Distances shown in FIG. 5, wherein “Yl” corresponds to the Yarrowia lipolytica Δ12 desaturase. Percent similarity and divergence are shown in the upper and lower triangles, respectively. Thus, the Y. lipolytica Δ12 desaturase was at least 53% identical to the other Δ12 desaturase homologs (having maximal identity to the A. nidulans sequence (An2)).

Example 4 Expression of Yarrowia lipolytica Δ12 Desaturase ORF Under the Control of a Heterologous Yarrowia Promoter

The present Example describes the expression of the Δ12 desaturase ORF in a chimeric gene under the control of a heterologous (non-Δ12 desaturase) Yarrowia promoter to complement the Δ12 desaturase-disrupted mutant and enable the overproduction of LA in the wildtype strain.

Expression of Y. lipolytica Δ12 Desaturase in Yarrowia lipolytica.

The ORF encoding the Y. lipolytica Δ12 desaturase was PCR amplified using upper primer P147 (SEQ ID NO:33) and lower primer P148 (SEQ ID NO:34) from the genomic DNA of Y. lipolytica ATCC #76982. The correct sized (1260 bp) fragment was isolated, purified, digested with Nco I and Not I and cloned into NcoI-Not I cut pY5-13 vector (Example 1), such that the gene was under the control of the TEF promoter. Correct transformants were confirmed by miniprep analysis and the resultant plasmid was designated pY25-d12d.

Plasmids pY5-13 (the “control”) and pY25-d12d were each individually transformed into Y. lipolytica ATCC #76982 wild-type (WT) and d12d-disrupted strains (Q-d12D, also referred to as “d12KO” in the Table below) and selected on Bio101 DOB/CSM-Leu plates.

Single colonies of transformants were grown up and GC analyzed as described in the General Methods. Results are shown in the Table below. Fatty acids are identified as 16:0, 16:1, 18:0, 18:1 (oleic acid) and 18:2 (LA); and the composition of each is presented as a % of the total fatty acids. “D12d SC” was calculated according to the following formula: ([18:2]/[18:1+18:2])*100 and represents percent substrate conversion. TABLE 8 Fatty Acid Composition (% Of Total Fatty Acids) % % % % % D12d Strain Plasmid 16:0 16:1 18:0 18:1 18:2 SC D12KO pY5-13 8 10 2 80 nd 0 D12KO pY25-d12d 11 8 2 34 45 57 WT pY5-13 10 10 1 32 47 59 WT pY25-d12d 12 7 2 21 59 74 *nd = not detectable

The results showed that the Δ12 desaturase promoter was equivalent in strength to the TEF promoter (57% substrate conversion in the d12KO strain expressing the Δ12 desaturase under the control of the TEF promoter, compared to 59% substrate conversion in the wild type strain expressing the Δ12 desaturase under the control of the native Δ12 desaturase promoter). On this basis, it is expected that the Δ12 desaturase promoter can be used for heterologous expression of other ORFs in Yarrowia.

Additionally, the results demonstrated that overexpression of the Δ12 desaturase in wild type cells resulted in even higher levels of LA production (18:2). Specifically, 74% substrate conversion was observed in the wildtype strain overexpressing the Δ12 desaturase under the control of the TEF promoter, as opposed to only 59% substrate conversion in the wild type strain. On the basis of these results, it would be expected that overexpression of the Δ12 desaturase, in combination of other genes for PUFA biosynthesis (e.g., a Δ6 desaturase, elongase, Δ5 desaturase, Δ17 desaturase), would result in higher production of ω-3 and/or ω-6 PUFAs. Additionally, it would be expected that disruption of the native Δ12 desaturase and expression of other genes for PUFA biosynthesis (e.g., a Δ6 desaturase, elongase, Δ5 desaturase, Δ17 desaturase) would result in production of “pure” ω-3 PUFAs, without co-synthesis of any ω-6 PUFAs.

Example 5 Selection of Δ6 Desaturase, Δ5 Desaturase, Δ17 Desaturase and High Affinity PUFA Elongase Genes For Expression in Yarrowia lipolytica

Prior to the introduction of specific genes encoding an ω-3 and/or ω-6 biosynthetic pathway into Yarrowia lipolytica containing a disrupted Δ12-desaturase (Example 8), it was necessary to confirm the functionality of heterologous Δ6 desaturase, elongase, Δ5 desaturase and Δ17 desaturase genes expressed in Yarrowia. This was accomplished by measuring the conversion efficiency of each wildtype protein in the alternate host. Specifically, a Mortierella alpina Δ5 desaturase, a M. alpina Δ6 desaturase, a Saprolegnia diclina Δ17 desaturase and a M. alpina high affinity PUFA elongase were separately expressed and screened for activity in substrate-feeding trials.

Construction of Expression Plasmids

In general, wildtype desaturase or elongase genes were either isolated by restriction digestion or amplified by PCR and inserted into appropriate vectors for expression. Each PCR amplification was carried out in a 50 μl total volume, comprising PCR buffer containing: 10 ng template, 10 mM KCl, 10 mM (NH₄)₂SO₄, 20 mM Tris-HCl (pH 8.75), 2 mM MgSO₄, 0.1% Triton X-100, 100 μg/mL BSA (final concentration), 200 μM each deoxyribonucleotide triphosphate, 10 pmole of each primer and 1 μl of PfuTurbo DNA polymerase (Stratagene, San Diego, Calif.). Amplification was carried out as follows (unless otherwise specified): initial denaturation at 95° C. for 3 min, followed by 35 cycles of the following: 95° C. for 1 min, 56° C. for 30 sec, 72° C. for 1 min. A final extension cycle of 72° C. for 10 min was carried out, followed by reaction termination at 4° C.

Wild Type Mortierella alpina (Accession #AF465281) Δ6 Desaturase

The 1384 bp NcoI/NotI fragment of pCGR5 (U.S. Pat. No. 5,968,809), which contains the M. alpina Δ6 desaturase gene (SEQ ID NO:36), was inserted into the NcoI/NotI sites of pY5-2 (Example 1) to generate pY54.

Wild Type Mortierella alpina (Accession #AF067654) Δ5 Desaturase

The M. alpina Δ5 desaturase gene (SEQ ID NO:38) was amplified by PCR using oligonucleotides YL11 and YL12 (SEQ ID NOs:40 and 41) as primers and plasmid pCGR-4 (U.S. Pat. No. 6,075,183) as template. PCR amplification was carried out as described above, with the exception that the elongation step was extended to 1.5 min (for cycles 1-35); The 1357 bp PCR product was digested with NcoI/NotI and ligated to NcoI/NotI-digested pY5-13 (described in Example 1) to generate pYMA5pb (FIG. 6).

Wild Type Saprolegnia diclina (ATCC #56851) Δ17 Desaturase

The Wild type Δ17 desaturase gene of S. diclina was amplified from plasmid pRSP19 (US 2003/0196217 A1) by PCR using oligonucleotides YL21A (SEQ ID NO:42) and YL22 (SEQ ID NO:43) as primers. The PCR products were digested with NcoI/PacI and then ligated to NcoI/PacI-digested pY54 (FIG. 4; described in Example 1) to generate pYSD17.

Wild Type Mortierella alpina (Accession #AX464731) High Affinity Elongase

The 973 bp NotI fragment of pRPB2 (WO 00/12720), containing the coding region of a M. alpina high affinity PUFA elongase gene (SEQ ID NO:44), was inserted into the NotI site of pY5 (described in Example 1; FIGS. 3 and 4) to generate pY58.

Transformation of Yarrowia lipolytica

The plasmids pY54, pYMA5pb, pYSD17 and pY58 were transformed separately into Y. lipolytica ATCC# 76982 according to the method of Chen, D. C. et al. (Appl Microbiol Biotechnol. 48(2):232-235 (1997)), and as described in Example 2 (with the exception that a leucine auxotroph of Yarrowia was used for transformation and transformants were selected on minimal media plates lacking leucine).

Determination of Percent Substrate Conversion

Single colonies of transformant Y. lipolytica containing pY54, pYMA5pb, pYSD17 or pY58 were each grown in 3 mL minimal media (20 g/L glucose, 1.7 g/L yeast nitrogen base without amino acids, 1 g/L L-proline, 0.1 g/L L-adenine, 0.1 g/L L-lysine, pH 6.1) at 30° C. to an OD₆₀₀˜1.0. For substrate feeding, 100 μl of cells were then subcultured in 3 mL minimal media containing 10 μg of substrate for about 24 hr at 30° C. Cells were subsequently collected by centrifugation and the lipids were extracted as described in the General Methods. Fatty acid methyl esters were prepared by transesterification of the lipid extract. Percent substrate conversion was determined as: [product/(substrate+product)]*100.

Percent Substrate Conversion By M. alpina Δ6 Desaturase

The M. alpina Δ6 desaturase converts LA to GLA and/or ALA to STA. Y. lipolytica strains containing pY54 were grown as described above (no substrate feeding required) and lipids were analyzed. The results showed that Yarrowia strains with pY54 converted about 30% LA to GLA.

Percent Substrate Conversion By M. alpina Δ5 Desaturase

The Δ5 desaturase from M. alpina converts DGLA to ARA and/or ETA to EPA. Y. lipolytica containing pYMA5pb was grown from a single colony, subcultured in minimal media containing 10 μg of DGLA and then subjected to lipid analysis as described above. Yarrowia strains with pYMA5pb converted about 30% of intracellular DGLA to ARA.

Percent Substrate Conversion By S. diclina Δ17 Desaturase

The S. diclina Δ17 desaturase converts ARA to EPA and/or DGLA to ETA. Y. lipolytica strains containing pYSD17 were grown from single colonies, subcultured in minimal media containing 10 μg of ARA and subjected to lipid analysis as described above. The results of the ARA feeding experiments showed that Yarrowia strains with pYSD17 converted about 23% of intracellular ARA to EPA.

Percent Substrate Conversion of Wild Type M. alpina High Affinity Elongase

The M. alpina high affinity PUFA elongase converts GLA to DGLA, STA to ETA, and/or EPA to DPA. Y. lipolytica strains containing pY58 were grown from single colonies, subcultured in minimal media containing 10 μg of GLA and subjected to lipid analysis as described above. The results of the GLA feeding experiments showed that Yarrowia strains with pY58 converted about 30% of intracellular GLA to DGLA.

Example 6 Synthesis and Expression of a Codon-Optimized Δ17 Desaturase Gene in Yarrowia lipolytica

Based on the results of Example 5, genes encoding Δ6 desaturase, elongase and Δ5 desaturase activies were available that each enabled ˜30% substrate conversion in Yarrowia lipolytica. The Δ17 desaturase from S. diclina, however, had a maximum conversion efficiency of only 23%. Thus, a codon-optimized Δ17 desaturase gene was designed, based on the Saprolegnia diclina DNA sequence (SEQ ID NO:35), according to the Yarrowia codon usage pattern, the consensus sequence around the ‘ATG’ translation initiation codon and the general rules of RNA stability (Guhaniyogi, G. and J. Brewer, Gene 265(1-2):11-23 (2001)).

In addition to modification to the translation initiation site, 127 bp of the 1077 bp coding region, comprising 117 codons, were codon-optimized. A comparison between this codon-optimized DNA sequence (SEQ ID NO:46) and the S. diclina Δ17 desaturase gene DNA sequence (SEQ ID NO:35) is shown in FIG. 7, wherein nucleotides in bold text correspond to nucleotides that were modified in the codon-optimized gene. None of the modifications in the codon-optimized gene changed the amino acid sequence of the encoded protein (SEQ ID NO:47).

The synthetic, codon-optimized Δ17 desaturase was suitable for expression with other genes for PUFA biosynthesis, to test the hypothesis of whether expression in a Yarrowia lipolytica host having its native Δ12 desaturase disrupted would result in production of “pure” ω-3 PUFAs, without co-synthesis of any ω-6 PUFAs (infra, Example 8).

Determining the Preferred Codon Usage in Yarrowia lipolytica

Approximately 100 genes of Y. lipolytica were found in the National Center for Biotechnology Information public database. The coding regions of these genes, comprising 121,167 bp, were translated by the Editseq program of DNAStar to the corresponding 40,389 amino acids and were tabulated to determine the Y. lipolytica codon usage profile shown in Table 9. The column titled “No.” refers to the number of times a given codon encodes a particular amino acid in the sample of 40,389 amino acids. The column titled “%” refers to the frequency that a given codon encodes a particular amino acid. Entries shown in bold text represent the codons favored in Yarrowia lipolytica. TABLE 9 Codon Usage In Yarrowia lipolytica Amino Amino Condon Acid No. % Condon Acid No. % GCA Ala (A) 359 11.4 AAA Lys (K) 344 14.8 GCC Ala (A) 1523 48.1 AAG Lys (K) 1987 85.2 GCG Ala (A) 256 8.1 AUG Met (M) 1002 100 GCU Ala (A) 1023 32.3 UUC Phe (F) 996 61.1 AGA Arg (R) 263 13.2 UUU Phe (F) 621 38.9 AGG Arg (R) 91 4.6 CCA Pro (P) 207 9.6 CGA Arg (R) 1133 56.8 CCC Pro (P) 1125 52.0 CGC Arg (R) 108 5.4 CCG Pro (P) 176 8.2 CGG Arg (R) 209 1.0 CCU Pro P 655 30.2 CGU Arg (R) 189 9.5 AGC Ser (S) 335 11.3 AAC Ans (N) 1336 84.0 AGU Ser (S) 201 6.8 AAU Ans (N) 255 16.0 UCA Ser (S) 221 7.5 GAC Asp (D) 1602 66.8 UCC Ser (S) 930 31.5 GAU Asp (D) 795 33.2 UCG Ser (S) 488 16.5 UGC Cys (C) 268 53.2 UCU Ser (S) 779 26.4 UGU Cys (C) 236 46.8 UAA Term 38 46.9 CAA Gln (Q) 307 17.0 UAG Term 30 37.0 CAG Gln (Q) 1490 83.0 UGA Term 13 16.1 GAA Glu (E) 566 23.0 ACA Thr (T) 306 12.7 GAG Glu (E) 1893 77.0 ACC Thr (T) 1245 51.6 GGA Gly (G) 856 29.7 ACG Thr (T) 269 11.1 GGC Gly (G) 986 34.2 ACU Thr (T) 595 24.6 GGG Gly (G) 148 5.1 UGG Trp (W) 488 100 GGU Gly (G) 893 31.0 UAC Tyr (Y) 988 83.2 CAC His (H) 618 65.5 UAU Tyr (Y) 200 16.8 CAU His H 326 34.5 GUA Val (V) 118 4.2 AUA Ile (I) 42 2.1 GUC Val (V) 1052 37.3 AUC Ile (I) 1106 53.7 GUG Val (V) 948 33.6 AUU Ile (I) 910 44.2 GUU Val (V) 703 24.9 CUA Leu (L) 166 4.7 CUC Leu (L) 1029 29.1 CUG Leu (L) 1379 38.9 CUU Leu (L) 591 16.7 UUA Leu (L) 54 1.5 UUG Leu (L) 323 9.1

For further optimization of gene expression in Y. lipolytica, the consensus sequence around the ‘ATG’ initiation codon of 79 genes was examined. In FIG. 8, the first ‘A’ of the underlined ATG translation codon is considered to be +1. Seventy seven percent of the genes analyzed had an ‘A’ in the −3 position, indicating a strong preference for ‘A’ at this position. There was also preference for ‘A’ or ‘C’ at the −4, −2 and −1 positions, an ‘A’, ‘C’ or ‘T’ at position +5, and a ‘G’ or ‘C’ at position +6. Thus, the preferred consensus sequence of the codon-optimized translation initiation site for optimal expression of genes in Y. lipolytica is ‘MAMMATGNHS’ (SEQ ID NO:130), wherein the nucleic acid degeneracy code used is as follows: M=A/C; S=C/G; H=A/C/T; and N=A/C/G/T.

In Vitro Synthesis of a Codon-Optimized Gene

The method used to synthesize the codon-optimized Δ17 desaturase gene is illustrated in FIG. 9. First, eleven pairs of oligonucleotides were designed to extend the entire length of the codon-optimized coding region of the S. diclina Δ17 desaturase gene (e.g., D17-1A, D17-1B, D17-2A, D17-2B, D17-3A, D17-3B, D17-4A, D17-4B, D17-5A, D17-5B, D17-6A, D17-6B, D17-7A, D17-7B, D17-8A, D17-8B, D17-9A, D17-9B, D17-10A, D17-10B, D17-11A and D17-11B, corresponding to SEQ ID NOs:48-69). Each pair of sense (A) and anti-sense (B) oligonucleotides were complementary, with the exception of a 4 bp overhang at each 5′-end. Additionally, primers D17-1A, D17-4B, D17-5A, D17-8A and D17-8B also introduced NcoI, BglII and SalI restriction sites for subsequent subcloning, respectively.

100 ng of each oligonucleotide was phosphorylated at 37° C. for 1 hr in a volume of 20 μl containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 10 mM DTT, 0.5 mM spermidine, 0.5 mM ATP and 10 U of T4 polynucleotide kinase. Each pair of sense and antisense oligonucleotides was mixed and annealed in a thermocycler using the following parameters: 95° C. (2 min), 85° C. (2 min), 65° C. (15 min), 37° C. (15 min), 24° C. (15 min) and 4° C. (15 min). Thus, D17-1A (SEQ ID NO:48) was annealed to D17-1B (SEQ ID NO:49) to produce the double-stranded product “D17-1AB”. Similarly, D17-2A (SEQ ID NO:50) was annealed to D17-2B (SEQ ID NO:51) to produce the double-stranded product “D17-2AB”, etc.

Three separate pools of annealed, double-stranded oligonucleotides were then ligated together, as shown below:

Pool 1: comprised D17-1AB, D17-2AB, D17-3AB and D17-4AB;

Pool 2: comprised D17-5AB, D17-6AB, D17-7AB and D17-8AB; and

Pool 3: comprised D17-9AB, D17-10AB and D17-11AB.

Each pool of annealed oligonucleotides was mixed in a volume of 20 μl with 10 U of T4 DNA ligase and the ligation reaction was incubated overnight at 16° C.

The product of each ligation reaction was then amplified by PCR. Specifically, using the ligated “Pool 1” mixture (i.e., D17-1AB, D17-2AB, D17-3AB, and D17-4AB) as template, and oligonucleotides D17-1 (SEQ ID NO:70) and D17-4R (SEQ ID NO:71) as primers, the first portion of the codon-optimized Δ17 desaturase gene was amplified by PCR. The PCR amplification was carried out in a 50 μl total volume, comprising PCR buffer containing 10 mM KCl, 10 mM (NH₄)₂SO₄, 20 mM Tris-HCl (pH 8.75), 2 mM MgSO₄, 0.1% Triton X-100, 100 μg/mL BSA (final concentration), 200 μM each deoxyribonucleotide triphosphate, 10 pmole of each primer and 1 μl of PfuTurbo DNA polymerase (Stratagene, San Diego, Calif.). Amplification was carried out as follows: initial denaturation at 95° C. for 3 min, followed by 35 cycles of the following: 95° C. for 1 min, 56° C. for 30 sec, 72° C. for 40 sec. A final extension cycle of 72° C. for 10 min was carried out, followed by reaction termination at 4° C. The 430 bp PCR fragment was subcloned into the pGEM-T easy vector (Promega) to generate pT17(1-4).

Using the ligated “Pool 2” mixture (i.e., D17-5AB, D17-6AB, D17-7AB and D17-8AB) as template, and oligonucleotides D17-5 (SEQ ID NO:72) and D17-8D (SEQ ID NO:73) as primers, the second portion of the codon-optimized Δ17 desaturase gene was amplified similarly by PCR and cloned into pGEM-T-easy vector to generate pT17(5-8). Finally, using the “Pool 3” ligation mixture (i.e., D17-9AB, D17-10AB and D17-11AB) as template, and oligonucleotides D17-8U (SEQ ID NO:74) and D17-11 (SEQ ID NO:75) as primers, the third portion of the codon-optimized Δ17 desaturase gene was amplified similarly by PCR and cloned into pGEM-T-easy vector to generate pT17(9-11).

E. coli was transformed separately with pT17(1-4), pT17(5-8) and pT17(9-11) and the plasmid DNA was isolated from ampicillin-resistant transformants. Plasmid DNA was purified and digested with the appropriate restriction endonucleases to liberate the 420 bp NcoI/BglII fragment of pT17(14), the 400 bp BglII/SalI fragment of pT17(5-8) and the 300 bp SalI/NotI fragment of pT17(9-11). These fragments were then combined, ligated together and used as template for amplification of the entire synthetic codon-optimized Δ17 desaturase gene using D17-1 (SEQ ID NO:70) and D17-11 (SEQ ID NO:75) as primers. The PCR amplification was carried out in a 50 μl total volume, using the conditions described above for each portion of the Δ17 desaturase gene and the thermocycling program as follows: initial denaturation at 95° C. for 3 min, followed by 35 cycles of the following: 95° C. for 1 min, 56° C. for 30 sec, 72° C. for 1.1 min. A final extension cycle of 72° C. for 10 min was carried out, followed by reaction termination at 4° C. This generated a 1.1 kB PCR product.

Construction of Plasmid pYSD17S Containing the Codon-Optimized Δ17 Desaturase

The 1.1 kB PCR product comprising the entire synthetic Δ17-desaturase was digested with NcoI/NotI and subcloned into NcoI/NotI-digested pY5-13 (Example 1) to generate pYSD17S (FIG. 10A).

As an additional “control”, to compare the efficiency of the wild type and synthetic genes in Yarrowia, the AT-rich PacI site in pYSD17 (comprising the wild-type gene; described in Example 5) was eliminated by site-directed mutagenesis using YL53 (SEQ ID NO:76) and YL54 (SEQ ID NO:77) as primers to generate pYSD17M (FIG. 10B).

Transformation of Yarrowia lipolytica with the Codon-Optimized Δ17 Desaturase Gene

Plasmids containing the wildtype and codon-optimized Δ17 desaturase were transformed separately into Y. lipolytica ATCC #76982 according to the methods described above in Example 5. Using this technique, transformants were obtained that contained the following plasmids: TABLE 10 Summary Of Plasmids In Transformant Yarrowia Plasmid Description pYSD17 wildtype Δ17 desaturase pYSD17M wildtype Δ17 desaturase, minus AT-rich PacI site pYSD17S codon-optimized Δ17 desaturase Percent Substrate Conversion with the Codon-Optimized Δ17 Desaturase Gene

Δ17 desaturase converts ARA to EPA (see FIG. 2). The percent substrate conversion ([product]/[substrate+product]*100) of the wildtype and codon-optimized Δ17 desaturase genes was determined in Yarrowia lipolytica containing each alternate plasmid construct, using the methodology described in Example 5.

The results of the ARA feeding experiments showed that Yarrowia strains with control plasmids pYSD17 or pYSD17M converted about 23% of intracellular ARA to EPA (FIG. 11A) while those containing the codon-optimized Δ17 desaturase gene within pYSD17S converted about 45% of intracellular ARA to EPA (FIG. 11B). Thus, Yarrowia containing the codon-optimized Δ17 desaturase converted about 2-fold more ARA than the strains containing the wild type S. diclina gene.

Example 7 Construction of Plasmids Suitable for the Coordinate Expression of Multiple Omega Fatty Acid Biosynthesis Genes in Yarrowia lipolytica

A variety of expression plasmids were constructed to produce a construct comprising a Δ6 desaturase, elongase, Δ5 desaturase, and Δ17 desaturase that would be suitable to integrate into the Y. lipolytica genome. Expression of this construct was necessary to test the hypothesize that “pure” ω-3 PUFAs, without co-synthesis of any ω-6 PUFAs, could be produced in a Y. lipolytica host containing a disrupted native Δ12 desaturase (infra, Example 8).

Construction of Plasmid pY24

Plasmid pY24 (FIG. 12) was a parent vector for construction of expression cassettes suitable for integration into the genome of Yarrowia lipolytica. pY24 was constructed as follows:

Using oligonucleotides KU5 and KU3 (SEQ ID NOs:78 and 79) as primers and Yarrowia genomic DNA as template, a 1.7 kB DNA fragment (SEQ ID NO:80) containing the Yarrowia URA3 gene was PCR amplified. The PCR amplification was carried out in a 50 μl total volume containing: 100 ng Yarrowia genomic DNA, PCR buffer containing 10 mM KCl, 10 mM (NH₄)₂SO₄, 20 mM Tris-HCl (pH 8.75), 2 mM MgSO₄, 0.1% Triton X-100, 100 μg/mL BSA (final concentration), 200 μM each deoxyribonucleotide triphosphate, 10 pmole of each primer and 1 μl of PfuTurbo DNA polymerase (Stratagene, San Diego, Calif.). Amplification was carried out as follows: initial denaturation at 95° C. for 3 min, followed by 35 cycles of the following: 95° C. for 1 min, 56° C. for 30 sec, 72° C. for 2 min. A final extension cycle of 72° C. for 10 min was carried out, followed by reaction termination at 4° C. The PCR product was inserted into pGEM-T easy vector (Promega, Madison, Wis.) to generate pGYUM.

Using oligonucleotides KI5 and KI3 (SEQ ID NOs:82 and 83), a 1.1 kB DNA fragment (SEQ ID NO:84) containing the conjugase gene (or “imp H8”) of Impatients balsama (clone ids.pk0001.h8; E. I. du Pont de Nemours and Company, Inc., Wilmington, Del.) was PCR amplified. The PCR amplification was carried out using the components described above, with the exception that 10 ng plasmid DNA of ids.pk0001.h8 was used as template. Amplification was carried out as follows: initial denaturation at 95° C. for 3 min, followed by 35 cycles of the following: 95° C. for 1.5 min, 56° C. for 30 sec, 72° C. for 1.2 min. A final extension cycle of 72° C. for 10 min was carried out, followed by reaction termination at 4° C. The PCR products were digested with NotI, and then inserted into the NotI site of pY5 (FIG. 3) to generate pY9.

Using oligonucleotides KTI5 and KTI3 (SEQ ID NOs:86 and 87), a 1.7 kB DNA fragment (SEQ ID NO:88) containing the TEF::IMP H8::XPR chimeric gene of pY9 was PCR amplified. The PCR amplification was carried out as described above, with the exception that 10 ng plasmid DNA of pGYUM was used as template. Amplification was carried out as follows: initial denaturation at 95° C. for 3 min, followed by 35 cycles of the following: 95° C. for 1 min, 56° C. for 30 sec, 72° C. for 2 min. A final extension cycle of 72° C. for 1-0 min was carried out, followed by reaction termination at 4° C. The PCR products were inserted into PCR-Script (Stratagene) to generate pY9R. The 1.7 kB Xho/EcoRV fragment of pY9R was exchanged with the XhoI/EcoRV fragment of pGYUM to generate pY21.

Using oligonucleotides KH5 and KH3 (SEQ ID NOs:90 and 91) as primers and genomic DNA of KS65 as template, a 1 kB DNA fragment (SEQ ID NO:92) containing the E. coli hygromycin resistance gene (“HPT”; Kaster, K. R., et al., Nucleic Acids Res. 11:6895-6911 (1983)) was PCR amplified. The PCR amplification was carried out in a 50 μl total volume using the components described above, with the exception that 10 ng plasmid DNA of ids.pk0001.h8 was used as template. Amplification was carried out as follows: initial denaturation at 95° C. for 3 min, followed by 35 cycles of the following: 95° C. for 1 min, 56° C. for 30 sec, 72° C. for 1.2 min. A final extension cycle of 72° C. for 10 min was carried out, followed by reaction termination at 4° C. The PCR products were digested with NotI and then inserted into the NotI site of pY5 (FIG. 3) to generate pTHPT-1.

Using oligonucleotides KTH5 and KTH3 (SEQ ID NOs:94 and 95) as primers and pTHPT-1 plasmid DNA as template, a 1.6 kB DNA fragment (SEQ ID NO:96) containing the TEF::HPT::XPR fusion gene was amplified as described above. The PCR products were digested with BglII and then inserted into pY21 to generate pY24.

Construction of pY24-4

Plasmid pY24 (FIG. 12) was used for construction of expression cassettes suitable for integration into the Y. lipolytica genome. The 401 bp of 5′-sequence (SEQ ID NO:98) and 568 bp of 3′-sequence (SEQ ID NO:99) from the Yarrowia lipolytica URA3 gene in pY24 plasmid were used to direct integration of expression cassettes into the Ura loci of the Yarrowia genome. Two chimeric genes (TEF::HPT::XPR and TEF::IMP H8::XPR) were first removed from pY24 by digestion with BamHI and self-ligation to generate pY24-1. PacI and BsiWI sites were introduced into pY24-1 by site-directed mutagenesis using YL63 and YL64 (SEQ ID NOs:100 and 101) and YL65 and YL66 (SEQ ID NOs:102 and 103) primer pairs, respectively, to generate pY24-4.

Construction of an Integration Vector for Expression of Δ5 Desaturase

The 4261 bp PacI/BsiWI fragment of pYMA5pb (comprising the M. alpina Δ5 desaturase gene; described in Example 5) was ligated into the PacI/BsiWI sites of pY24-4 (FIG. 12) to generate pYZM5 (FIG. 6). HindIII and ClaI sites were introduced into pYZM5 by site-directed mutagenesis using primer pairs YL81 and YL82 (SEQ ID NOs:104 and 105) and YL83 and YL84 (SEQ ID NOs:106 and 107), respectively, to generate pYZM5CH (FIG. 6). A PmeI site was introduced into pYZM5CH by site-directed mutagenesis using YL105 and YL106 (SEQ ID NOs:108 and 109) as primers to generate pYZM5CHPP. An AscI site was introduced into pYZM5CHPP by site-directed mutagenesis using YL119 and YL120 (SEQ ID NOs:110 and 111) as primers to generate pYZM5CHPPA (FIG. 6).

To optimize the integration vector, 440 bp of 5′-non-coding DNA sequence upstream from the Yarrowia lipolytica URA3 gene (SEQ ID NO:114) was amplified by PCR using YL121 and YL122 (SEQ ID NOs:112 and 113) as primers. The PCR product was digested with AscI and BsiWI and then exchanged with the AscI/BsiWI fragment of pYZM5CHPPA (FIGS. 6 and 13) to generate pYZM5UPA (FIG. 13). An AscI site was introduced into pYZM5UPA by site-directed mutagenesis using oligonucleotides YL114 and YL115 (SEQ ID NOs:115 and 116) to generate pYZV5. In order to reduce the size of the 3′-non-coding region of the URA3 gene in pYZV5, a second PacI site was introduced into the middle of this region by site-directed mutagenesis using oligonucleotides YL114 and YL115 (described above) to generate pYZV5P. The PacI fragment of pYZV5P was excised by digestion with PacI and religation to generate pYZV16 (FIG. 13). Digestion of pYZV16 with AscI liberates a 5.2 kB DNA fragment (SEQ ID NO:117) suitable for integration and expression of the Δ5 desaturase gene (“MAD5”) in the Y. lipolytica genome.

Construction of an Integration Vector for Expression of the High Affinity Elongase and Δ5 Desaturase

BsiWI and HindIII sites were introduced into pY58 (containing the coding region of the M. alpina high affinity PUFA elongase; described in Example 5) by site-directed mutagenesis using YL61 and YL62 (SEQ ID NOs:17 and 18) and YL69 and YL70 (SEQ ID NOs:118 and 119) primer pairs, respectively, to generate pY58BH (FIG. 14; elongase gene labeled as “EL”). The 1.7 kB BsiWI/HindIII fragment of pY58BH, which contains the TEF::EL::XPR chimeric gene, was ligated into the BsiWI/HindIII site of pYZM5CHPP (construction described in FIG. 6) to generate pYZM5EL (FIG. 14). This plasmid is suitable for integration and coordinate expression of the M. alpina Δ5 desaturase and high affinity PUFA elongase genes in Y. lipolytica.

Construction of an Integration Vector for Expression of the Δ6 Desaturase, High Affinity Elongase and Δ5 Desaturase

PacI and ClaI sites were introduced into pY54 (containing the M. alpina Δ6 desaturase; described in Example 5) by site-directed mutagenesis using YL77 and YL78 (SEQ ID NOs:120 and 121) and YL79A and YL80A (SEQ ID NOs:122 and 123) primer pairs, respectively, to generate pY54PC (FIG. 14; Δ6 desaturase gene labeled as “MAD6”). The 2 kB ClaI/PacI DNA fragment of pY54PC, which contains the TEF::MAD6::XPR chimeric gene, was ligated into the ClaI/PacI sites of pYZM5EL to generate pYZM5EL6 (FIG. 14). This plasmid is suitable for integration and coordinate expression of the M. alpina Δ6 desaturase, Δ5 desaturase and high affinity PUFA elongase genes in the Y. lipolytica genome.

Construction of a DNA Fragment Suitable for Integration into the Yarrowia Genome, for Expression of the Δ6 Desaturase, PUFA Elongase and Δ5 Desaturase

The plasmid pYZV16 (construction described in FIG. 13) was used for construction of plasmids containing multiple expression cassettes.

First, the 3.5 kB BsiWI/PacI fragment of pYZV16 was ligated to the 7.9 kB BsiWI/PacI fragment of pYZM5EL6 (construction described in FIG. 14) to generate pYZV5EL6 (FIG. 15). Digestion of pYZV5EL6 with AscI liberates a 8.9 kB DNA fragment (SEQ ID NO:124) suitable for integration and coordinate expression of the Δ6 desaturase, PUFA elongase and Δ5 desaturase genes in the Y. lipolytica genome.

Construction of a DNA Fragment Suitable for Integration into the Yarrowia Genome, for Expression of the Δ6 Desaturase, PUFA Elongase, Δ5 Desaturase and Δ17 Desaturase

A synthetic S. diclina Δ17 desaturase gene was inserted into the NcoI/NotI sites of pY5-13 to generate pYSD17S (FIG. 1A). ClaI and PmeI sites were introduced into pYSD17S by site-directed mutagenesis using YL101 and YL102 (SEQ ID NOs:125 and 126) and YL103 and YL104 (SEQ ID NOs:127 and 128) primer pairs, respectively, to generate pYSD17SPC (FIG. 15).

The 347 bp ClaI/PmeI fragment of pYZV5EL6 (FIG. 15) was exchanged with the 1760 bp ClaI/PmeI fragment from pYSD17SPC containing the Δ17 desaturase expression cassette to generate pYZV5E6/17. Digestion of pYZV5E6/17 with AscI liberates a 10.3 kB DNA fragment (SEQ ID NO:129) suitable for integration and coordinate expression of the Δ6 desaturase, PUFA elongase, Δ5 desaturase and Δ17 desaturase genes in the Y. lipolytica genome.

Example 8 Use of Δ12 Desaturase Disrupted Strains for the Production of Pure Omega-3 Fatty Acids by Substrate Feeding

The present Example describes the utility of a Δ12 desaturase-disrupted Yarrowia lipolytica host strain containing appropriate heterologous genes (e.g., a Δ6 desaturase, elongase, Δ5 desaturase, Δ17 desaturase, as described in Example 7) for the production of ω-3 PUFAs, without co-synthesis of any ω-6 PUFAs. Feeding studies were performed with ALA as the substrate. The results demonstrate that it is possible to produce ω-3 PUFAs in the absence of ω-6 PUFAs.

Feeding Studies

Wildtype Yarrowia lipolytica ATCC #76982 was transformed with an integrating 10.3 kB DNA fragment (SEQ ID NO:129) containing a Δ6 desaturase, PUFA elongase, Δ5 desaturase and Δ17 desaturase (from Example 7). This resulted in creation of strain “WT+4G”. Then, the Δ12 desaturase was disrupted in strain WT+4G, as described in Example 2. This resulted in creation of strain “D12KO+4G”.

Cells from each of the four strains listed below in Table 11 (100 μl) were grown in 3 mL minimal media containing no substrate addition, 10 μg of LA, 10 ug ALA, or 5 ug each of LA and ALA for about 24 hr at 30° C. TABLE 11 Description Of Strains Tested In The Feeding Studies Strain Designation Strain Description Example WT wild-type Yarrowia lipolytica ATCC #76982 — WT + 4G wild-type Yarrowia lipolytica, containing a 8 Δ6 desaturase, PUFA elongase, Δ5 desaturase and Δ17-desaturase D12KO Δ12 desaturase-disrupted Yarrowia 2 lipolytica D12KO + 4G Δ12 desaturase-disrupted Yarrowia 8 lipolytica, containing a Δ6 desaturase, PUFA elongase, Δ5 desaturase and Δ17 desaturase

Fatty acid composition was determined by direct transesterification, as described in the General Methods. The fatty acid profile of each of the strains grown with no substrate addition, 10 μg of LA, 10 ug ALA, or 5 ug each of LA and ALA are shown below in Table 12. Fatty acids are identified as 16:0, 16:1, 18:0, 18:1 (oleic acid), 18:2 (LA), GLA, DGLA, ALA, and STA. The composition of each is presented as a % of the total fatty acids. TABLE 12 Fatty Acid Composition (% Of Total Fatty Acids) Strain FA feed 16:0 16:1 18:0 18:1 18:2 GLA DGLA ALA STA WT None 10 6 8 50 21 nd nd nd nd WT LA 11 3 6 30 47 nd nd nd nd WT ALA 9 3 4 29 5 nd nd 48 nd D12KO None 10 7 8 68 nd nd nd nd nd D12KO LA 9 4 5 26 53 nd nd nd nd D12KO ALA 10 4 6 41 nd nd nd 35 nd WT + 4G None 11 6 7 57 6 5 0.9 nd nd WT + 4G LA 11 3 6 32 31 9 1.0 nd nd WT + 4G ALA 9 3 5 31 2 1 0.2 40 4 WT + 4G LA + A 6 1 2 10 33 4 0.3 39 2 D12KO + 4G None 9 6 8 69 nd nd nd nd nd D12KO + 4G LA 8 2 6 24 45 10 1.0 nd nd D12KO + 4G ALA 8 5 5 45 nd nd nd 27 4 D12KO + 4G LA + A 7 2 4 14 26 4 0.3 37 3 *nd = not detectable

The results showed that feeding ALA to D12 KO cells resulted in the production of only ω-3 fatty acids (i.e., ALA and STA), without biosynthesis of any ω-6 fatty acids (i.e., GLA or DGLA). 

1-12. (canceled)
 13. A method for the production of linoleic acid comprising: a) providing a microbial host cell comprising: (i) a chimeric gene encoding a Δ12 desaturase polypeptide selected from the group consisting of: (a) an isolated nucleic acid molecule encoding the amino acid sequence as set forth in SEQ ID NO:24; (b) an isolated nucleic acid molecule that hybridizes with (a) under the following hybridization conditions: 0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS; and (ii) a source of desaturase substrate consisting of oleic acid; b) growing the microbial host cell of step (a) under conditions wherein the gene encoding a Δ12 desaturase polypeptide is expressed and the oleic acid is converted to linoleic acid; and c) optionally recovering the linoleic acid of step (b). 14-16. (canceled)
 17. A method according to claim 13 wherein the host cell is a yeast cell.
 18. A method according to claim 17 wherein the yeast cell is an oleaginous yeast selected from the group consisting of Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces.
 19. A method according to claim 18 wherein the oleaginous yeast is Yarrowia sp.
 20. A method according to claim 19 selected from the group consisting of Yarrowia lipolytica ATCC #20362, Yarrowia lipolytica ATCC #8862, Yarrowia lipolytica ATCC #18944, Yarrowia lipolytica ATCC #76982 and Yarrowia lipolytica LGAM S(7)1.
 21. A method for modulating the biosynthesis of ω-3 or ω-6 fatty acids in a microbial host cell comprising: a) providing a microbial host cell comprising a functional ω-3/ω-6 fatty acid biosynthetic pathway; b) over-expressing a Δ12 desaturase gene in the host cell of (a); whereby the biosynthesis of ω-3 or ω-6 fatty acids is modulated.
 22. A method according to claims 21 wherein the host cell is an oleaginous yeast.
 23. A method according to claim 22 wherein the host cell is Yarrowia and the Δ12 desaturase gene encodes the Δ12 desaturase enzyme as set forth in SEQ ID NO:24.
 24. A method according to claim 21 wherein the Δ12 desaturase gene is over-expressed on a multicopy plasmid.
 25. A method according to claim 21 wherein Δ12 desaturase gene is operably linked to an inducible or regulated promoter. 26-28. (canceled) 